Inhibition of L-selectin and P-selection mediated binding using heparin

ABSTRACT

The present invention provides methods of inhibiting L-selectin and P-selectin mediated binding in a subject by administering heparin to the subject in an amount that does not produce substantial anticoagulant activity or undesirable bleeding in the subject. In addition, the invention provides methods of treating a subject having a pathology characterized, at least in part, by abnormal L-selectin or P-selectin mediated binding by administering heparin to the subject in an amount that results in attaining a concentration of less than about 0.2-0.4 units heparin per ml of plasma in the subject.

This application is based on, and claims the benefit of, U.S.Provisional Application No. 60/073,998, filed Feb. 9, 1998, and isincorporated herein by reference.

Each of the references cited herein, including the references numbered18, 25 and 57 to 200, is incorporated herein by reference.

This invention was made with government support under grant numbersCA38701 and HL23584 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to molecular biology and, morespecifically, to methods of modulating L-selectin and P-selectinmediated binding in a subject by administering heparin to the subject inan amount that does not produce substantial anticoagulation activity orundesirable bleeding.

2. Background Information

L-selectin, E-selectin and P-selectin mediate the initial adhesiveevents directing the homing of lymphocytes into lymphoid organs, as wellas the interactions of leukocytes and other inflammatory cells withendothelium at sites of inflammation. L-selectin is expressed onleukocytes, E-selectin is expressed on endothelium and P-selectin isexpressed on platelets and endothelium. The three selectins bind tospecific carbohydrate structures on opposing cells, for example,L-selectin binds to platelets and endothelium, whereas P-selectin andE-selectin bind to leukocytes.

Selectin adhesion is involved in disorders such as pathologicreperfusion injury, inflammatory disorders and autoimmune disorders.Selectin interactions also can mediate primary adhesive mechanismsinvolved in the metastasis of certain epithelial cancers. Thus,selectins are potential therapeutic targets for the treatment ofpathologies characterized by undesirable or abnormal interactionsmediated by selecting.

Much work has been directed to finding small carbohydrate molecules foruse as competitive inhibitors to block selectin mediated interactions.For example, the tetrasaccharide sialyl-Lewis^(x) (SLe^(x)) isrecognized by all three selectins, and is a component of many naturallyoccurring high affinity selectin ligands, for example, the myeloid cellligand for P-selectin, called PSGL-1. However, the interaction ofselectins with purified SLe^(x) is weak and SLe^(x) demonstrates littleselectivity among the selecting. Thus, while SLe^(x) and relatedstructures may provide some therapeutic use, they are limited in beingweak and nonselective inhibitors of selectin binding. Furthermore, theyare very expensive to produce in the quantities required for treatment.

Heparan sulfates (HS) are naturally occurring glycosaminoglycan (GAG)chains that have diverse biological functions, generally mediated bytheir ability to interact with growth factors, receptors and the like.Heparin, which is a heavily modified GAG, is a heterogeneous mixture oflong, unbranched carbohydrate chains consisting of repeatingdisaccharide units composed of uronic acids alternating with glucosamineresidues which can be extensively modified.

Due to its anticoagulant activity, heparin is used clinically as anantithrombotic agent for treating human subjects having a disorderresulting from the abnormal or undesirable activation of the bloodclotting cascade. Heparin's anticoagulant activity is the result of amodified pentasaccharide sequence that is present on certain heparinchains and binds to antithrombin III, a regulatory protein of theclotting cascade. Heparin chains that are longer than about 18saccharide units and that have the modified pentasaccharide sequence canenhance the ability of antithrombin III to bind to and inhibit thefunction of coagulation factors, thereby inhibiting the blood clottingcascade. Pharmaceutical preparations of heparin are enriched forantithrombin III binding chains, but also contain a mixture of othercomponents.

L-selectin and P-selectin interact with a variety of sulfated compoundsincluding heparan sulfates, porcine intestinal mucosal (PIM) heparin andits fragments. Although L-selectin and P-selectin similarly bind to HSchains and PIM-heparin, they are different from E-selectin, which failsto bind the HS chains or PIM-heparin. The observation that L-selectinbinding to endothelial cell HS chains is calcium dependent indicatesthat this interaction is similar to selectin's interaction with naturalligands. This suggestion is supported by the ability of small heparinfragments to compete with L-selectin and P-selectin binding to SLe^(x).

Pharmaceutical preparations of heparin are similar to crude commercialPIM-heparin. Thus, it might be expected that pharmaceutical heparinwould have been used for inhibiting the binding of L-selectin andP-selectin to ligands present on cells in humans. However, heparin hasnot been used for the purpose of inhibiting L-selectin and P-selectinbinding in humans because of concerns about potential undesirable sideeffects associated with its anticoagulant activity.

To address the problem of undesirable anticoagulant activity, lowmolecular weight (LMW) oligosaccharides, which are derived from heparin,but lack anticoagulant activity, have been prepared. When injected intomice, such LMW heparins can inhibit inflammation by binding toL-selectin and P-selectin. However, the cost of preparing LMW forms ofheparin combined with the cost of new product testing to obtain FDAapproval for use in humans is often prohibitive. In addition, it isunclear how efficacious the LMW heparins would be in inhibitingL-selectin and P-selectin binding when administered to a human subject.Thus, a need exists for developing methods of using readily availablepharmaceutical compositions to inhibit L-selectin and P-selectinmediated binding in a subject without producing undesirable sideeffects. The present invention satisfies this need and provides relatedadvantages as well.

SUMMARY OF THE INVENTION

The present invention provides a method of inhibiting L-selectin andP-selectin binding in a subject by administering to the subject anamount of heparin that does not produce substantial anticoagulantactivity or undesirable bleeding. Thus, the invention provides methodsof administering heparin in amounts that result in attaining aconcentration less than about 0.2-0.4 units heparin per ml of plasma inthe subject, such levels which inhibit L-selectin or P-selectin mediatedbinding in the subject. The invention further provides methods oftreating a subject having an L-selectin or P-selectin related pathologyby administering heparin in an amount that does not produce substantialanticoagulant activity or undesirable bleeding, for example, an amountthat results in a concentration less than about 0.2-0.4 units heparinper ml of plasma in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows that heparin sulfate obtained from endothelial cells bindsto L-selectin. FIG. 1B shows that heparin sulfate obtained fromendothelial cells does not bind to E-selectin. FIG. 1C shows thatheparin sulfate obtained from endothelial cells binds to P-selectin. Theelution profile of HS chain from an L-selectin, P-selectin or E-selectinaffinity column using buffer containing 5 mM EDTA is shown. Arrowsindicate fraction where EDTA elution began.

FIG. 2A show that the binding of heparin sulfate glycosaminoglycanchains (HS-GAG) by L-selectin, is calcium-dependent. FIG. 2B shows thatbinding of heparin sulfate glycosaminoglycan chains (HS-GAG) byP-selectin is not calcium-dependent. HS-GAG chains were applied eitherto an L-selectin or P-selectin column in the presence of 5 mM calcium(solid triangles) or of magnesium/EGTA (open circles). The elutionprofiles were obtained using 5 mM EDTA buffer for L-selectin and 20 mMEDTA buffer for P-selectin. Arrows as in FIG. 1.

FIG. 3 shows that the binding of the HS-GAG chains to P-selectin doesnot require exogenously added cations. HS-GAG in buffer lackingexogenously added cations was applied to a P-selectin column, washedwith the same buffer and eluted with 20 mM EDTA buffer. Arrows as inFIG. 1.

FIG. 4A shows that fractions of porcine intestinal mucosal (PIM) heparindo not bind to E-selectin. FIG. 4B shows that fractions of porcineintestinal mucosal (PIM) heparin can bind to L-selectin. FIG. 4C showsthat fractions of porcine intestinal mucosal (PIM) heparin can bind toP-selectin. PIM-Heparin was applied to L-selectin, P-selectin or anE-selectin affinity column; the column was washed and eluted with 2 mMEDTA, followed by 20 mM EDTA buffer.. Arrows as in FIG. 1.

FIG. 5A shows the results of experiments in which the binding ofPIM-heparin to L-selectin was shown to be calcium dependent. FIG. 5Bshows the results for PIM-heparin applied to a P-selectin affinitycolumn. In both of these experiments, the PIM-heparin was applied in amagnesium/EGTA buffer lacking exogenously added calcium. The columnswere and eluted with 2 mM EDTA followed by 20 mM EDTA buffer; thepercentage eluted in various peaks also is shown. Arrows as in FIG. 1.

FIGS. 6A-6D show the size dependence of porcine mucosal heparinfragments in binding to an L-selectin affinity column. FIG. 6A shows theresults for octasaccharides, while FIG. 6B shows the results fordecasaccharides, FIG. 6C shows the results for dodecasaccharides, andFIG. 6D shows the results for tetrasaccharides. In these experiments,the columns were eluted with 2 mM EDTA (“2”), followed by 20 mM EDTA(“20) buffer. Arrows as in FIG. 1.

FIGS. 7A-7D show the results of rechromatographing PIM-heparintetradecasaccharides on an L-selectin affinity column. FIG. 7A shows theresults for tetradecasaccharides that originally were unbound (Pool A),while FIG. 7B shows the results for tetradecasaccharides that wereslightly retarded (Pool B), FIG. 7C shows the results fortetradecasaccharides that were retarded (Pool C), and FIG. 7D shows theresults for tetradecasaccharides that were eluted with EDTA. Thetetradecasaccharides from each pool were eluted with 2 mM EDTA buffer.The horizontal bar above each chromatogram indicates where the pooloriginally eluted from the column. Arrows as in FIG. 1.

FIG. 8A shows the inhibitory properties of a pharmaceutical heparinpreparation (unfractionated; closed triangles) or LMW heparins (LOVENOX,open squares; FRAGMIN, closed circles) on the binding of L-selectin toimmobilized SLe* using ELISA inhibition experiments, while FIG. 8B showsthe inhibitory properties of a pharmaceutical heparin preparation(unfractionated; closed triangles) or LMW heparins (LOVENOX, opensquares; FRAGMIN, closed circles) on the binding of P-selectin toimmobilized SLe* using ELISA inhibition experiments. “Units/ml”indicates that concentration of heparin or LMW heparins in the reaction.

FIG. 9 shows the inhibition of HL-60 cell attachment to immobilizedL-selectin (open squares) or P-selectin (closed circles) by apharmaceutical heparin preparation (unfractionated). The horizontal barindicates the recommended plasma concentration range for therapeutic useof heparin as an anticoagulant in a human subject.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of inhibiting L-selectin orP-selectin mediated binding in a subject by administering to the subjectan amount of heparin that does not produce substantial anticoagulantactivity or undesirable bleeding in the subject. Heparin is usedclinically as an anticoagulant to reduce blood clotting in anindividual. Heparin has a narrow therapeutic window and is administeredto individuals for anticoagulant therapy in an amount sufficient toachieve levels of 0.2-0.4 units per ml of plasma (Ginsberg, New Engl. J.Med. 335:1816-1828 (1996)). Individuals administered heparin atconcentrations less than 0.2 units per ml of plasma are significantlymore likely to exhibit a recurrence of thrombosis, indicating thatadministration of an amount of heparin that results in a concentrationof less than about 0.2 units per ml of plasma generally is not optimalfor anticoagulant therapy of an active thrombus.

As disclosed herein, heparin, as formulated for clinical use, caninhibit the binding of P-selectin and L-selectin to their ligands.Significantly, approximately one-fifth to one-tenth of the amount ofpharmaceutical heparin that is needed for anticoagulant therapy inhumans effectively inhibits L-selectin and P-selectin mediated binding.Thus, the invention provides a means to inhibit L-selectin andP-selectin mediated binding in a subject by administering heparin in anamount that does not produce substantial anticoagulant activity orundesirable bleeding in the subject.

The amount of heparin administered to a subject to inhibit L-selectin orP-selectin binding is characterized in that it does not produceundesirable bleeding as a side effect, although it can produce mildanticoagulant activity. As a result, side effects such as bleedingcomplications that are associated with using heparin for anticoagulanttherapy are not a concern. The amount of heparin administered in amethod of the invention results in a concentration of less than about0.2 to 0.4 units heparin/ml plasma, generally about 0.1 to 0.2 unitsheparin/ml plasma, or about 0.05 to 0.1 units heparin/ml plasma, and canbe about 0.02 units heparin/ml plasma or less.

The invention additionally provides a method of inhibiting P-selectinmediated binding, but not L-selectin mediated binding, in a subject byadministering heparin in an amount that results in a concentration ofabout 0.02 to 0.05 units heparin/ml plasma, preferably about 0.005 to0.02 units heparin/ml plasma to the subject. For example, an amount ofheparin that results in about 0.002 units heparin/ml plasma can beadministered to a subject to inhibit P-selectin mediated binding,whereas such a concentration of heparin does not substantially inhibitL-selectin mediated binding.

Although the ability of heparin to inhibit L-selectin and P-selectinbinding has been established, its anticoagulant activity has preventedclinicians from using heparin to inhibit L-selectin and P-selectinbinding in a subject (Yednock et al., J. Cell Biol. 104:713-723 (1987);Skinner et al., Biochem. Biophys. Res. Commun. 164:1373-1379 (1989);Aruffo et al., Cell 67:35-44 (1991); Handa et al., Biochem. Biophys.Res. Commun. 181:1223-1230 (1991); Skinner et al., J. Biol. Chem.266:5371-5374 (1991); Needham and Schnaar, Proc. Natl. Acad. Sci. USA90:1359-1363 (1993); Nelson et al., Blood 82:3253-3258 (1993); Yuen, etal., J. Biol. Chem. 269:1595-1598 (1994); Mitsuoka et al., Biochem.Biophys. Res. Commun. 230:546-551 (1997); Yoshino et al., J. Med. Chem.40:455-462 (1997); Norgard-Sumnicht et al., Science 261:480-483 (1993)).Thus, molecules that can inhibit L-selectin and P-selectin bindingwithout exhibiting anticoagulant activity have been identified. Forexample, Bevilacqua et al. demonstrated that particular low molecularweight fractions of heparin which lack anticoagulant activity modulateselectin binding (U.S. Pat. No. 5,527,785, issued Jun. 18, 1996, whichis incorporated herein by reference). Bevilacqua et al. also used crudenonpharmaceutical grade heparin to inhibit selectin binding in vitro.However, Bevilacqua et al. stated that heparin compositions that containthe pentasaccharide antithrombin III binding sequence, which is presentin pharmaceutical heparin preparations, should not be used forinhibiting selectin mediated binding in a subject due to itsanticoagulant activity.

As disclosed herein, pharmaceutical preparations of heparin inhibitL-selectin and P-selectin mediated binding at concentrations less thanabout 0.2-0.4 units heparin per ml of plasma. This amount of heparin canproduce mild anticoagulant activity in humans, but usually does notproduce undesirable bleeding. For example, two separate lots ofunfractionated pharmaceutical heparin significantly inhibited L-selectinand P-selectin binding at concentrations less than the recommendedtherapeutic levels targeted for anticoagulation therapy in a subject. Inparticular, the concentration of pharmaceutical heparin needed toinhibit 50% of the binding (IC₅₀) of SLe^(x) to L-selectin andP-selectin was 0.07-0.08 units/ml and 0.01-0.02 units/ml, respectively(see Table 5, below). In addition, IC₅₀ values for pharmaceuticalheparin to inhibit attachment of HL-60 cells, which express the naturalselectin ligand PSGL-1, to immobilized selectins were 0.02-0.03 units/mlfor L-selectin and 0.003-0.01 units/ml for P-selectin (Table 5). Thus,clinically approved heparin compositions can inhibit L-selectin andP-selectin mediated binding in an amount that does not producesubstantial anticoagulant activity or undesirable bleeding in a subject.

The methods of the invention are useful in treating a subject having anL-selectin or P-selectin related pathology. Many pathological processesinvolve L-selectin and P-selectin (Table 1) and, as disclosed herein,can be treated by administering heparin in an amount that results in aconcentration of less than about 0.2-0.4 units/ml of plasma in asubject. Ischemia and reperfusion, for example, which cause significanttissue injury in clinical disorders such as stroke, myocardialinfarction, organ transplantation and organ hypoperfusion, involveL-selectin and P-selectin related processes (Seekamp et al., Am. J.Pathol. 144:592-598 (1994); Garcia-Criado et al., J. Am. Coll. Surgeons181:327-334 (1995); Moore et al., J. Appl. Physiol. 78:2245-2252 (1995);Mihelcic et al., Blood 84:2322-2328 (1994); Han et al., J. Immunol.155:4011-4015; Rabb et al., Am. J. Physiol. Renal Fluid ElectrolytePhysiol. 271:F408-F413 (1996); Buerke et al., J. Clin. Invest.93:1140-1148 (1994); Miura et al., Ann. Thorac. Sura. 62:1295-1300(1996); Flynn et al., Am. J. Physiol. Heart Circ. Physiol.271:H2086-H2096 (1996); Ma et al., Circulation 88:649-658 (1993); Buerkeet al., J. Pharmacol. Exp. Ther. 271:134-142 (1994); Lefer, Ann. Thorac.Surg. 60:773-777 (1995); Haught et al., Am. Heart J. 132:1-8 (1996);Turunen et al., J. Exp. Med. 182:1133-1141 (1995); Morikawa et al.,Stroke 27:951-955 (1996); Weiser et al., Shock. 5:402-407 (1996); Yamadaet al., Blood 86:3487-3492 (1995); Kubes et al., Am. J. Physiol. HeartCirc. Physiol. 267:H931-H937 (1994); Gibbs et al., Surgery 119:652-656(1996); Takada et al., J. Clin. Invest. 99:2682-2690 (1997); Zizzi etal.,

TABLE 1 TISSUE PATHOLOGY REFERENCE Pathological processes involvingL-selectin Lung Homing of eosinophils to asthmatic lungs (58, 59) Acutelung injury (60, 61, 61-64) Inflammation (65, 66) Ischemia-reperfusioninjury (67, 68) Sepsis-induced lung injury (69) Adult respitory distresssyndrome (ARDS) (57) Multi- Inflammatory injury (70, 70-72) systemIschemia-reperfusion injury (67) Immune HIV infection (73) Impairedprimary T-cell response (74) Common variable immunodefiency (CVID) (75)Chronic myelocytic leukemia (76) Primary Sjogren's syndrome (77)Inflamed extralymphoid tissues (78) Humoral immune response (in spleen)(79) Meningeal leukemia (80) Pancreas Nonobese diabetes (81-83)Skeletal; Rheumatoid arthritis (84) Connective Anklosing spondylitis(85) tissue System schlerosis (85) Vasculitis (85) MuscularIschemia-reperfusion injury (67) Liver Primary infection with Listeriamonocytogenes (86) Liver inflammation (87) Skin Cutaneous inflammation(88) Ischemia-reperfusion injury (89, 90) Systemic inflammatory responsesyndrome (SIRS) (91, 92) Gut Inflammatory injury (93) inflamedperitoneum (94) Colon carcinoma (95) Kidney Allograph rejection (25)Impaired granulocyte function in patients with (96) chronic hemodialysisRenal ischemia-reperfusion injury (97) Circulatory Inflamed/injuredvenular endothelium (98, 99) Ischemia-reperfusion injury (100-106) PMNleukocyte-induced vasocontraction and (107) endothelial dysfuntionsHemorrhagic shock injury (108) Peripheral arterial disease (109) Cardiactransplant rejection (110) Activated endothelium (111) CNS Pleocytosisin bacterial meningitis (112) Allergic encephalomyelitis (113)Memogoencephalitis (114) Ischemia reperfusion injury (113) Multiplesclerosis (MS) (116) Misc. Failure of tumor immunity (117) Plasmodiumfalciparium malaria (118) Increased soluble L-selectin in prematureinfants (119, 120) Burn injury (57) Hyperthyroidism (121) Pathologicalprocesses involving P-selectin Lung Acute lung injury (60, 63, 64,22-127) Reduction of allergic airway hyperresponsiveness (128)Ischemia-reperfusion injury (67) Inflammation induced by Streptococcuspneumonia (66) Hyperoxic lung injury (129) Small cell lung carcinoma(130) Adult respitory distress syndrome (ARDS) (57) Asthma (131)Pleurisy (132) Multi- Inflammatory injury (18, 72, 133) system Sepsisand organ failure due to severe trauma (134) Burn injury (57, 135)Ischemia-reperfusion injury (67) Immune Leukocyte adhesion syndrome,type II (LAD II) (136) Infection susceptibility (137) Primary Sjogren'sSyndrome (77) Arthus reaction (138) Lack of recruitment of Helper T-1cells to (139, 140) inflammatory sites Peripherial neutrophilia (141)Altered serum selectin levels in leukemia patients (142) Alteredhematopoiesis and infection susceptibility (137, 143) Severe trauma,sepsis, organ failure (134) Pancreas Nonobese diabetes (144) Skeletal;Rheumatoid arthritis (84, 145, 146) Connective tissue Muscular Skeletalmuscle ischemia-reperfusion injury (147) Injury and inflammation tostriated muscle (148) Liver Ischemia-reperfusion injury (149) Improvedliver function after hemorrhagic shock (150) Skin Cutaneous inflammation(88, 148, 151) Ear reperfusion injury (90, 152) Ischemia-reperfusioninjury in dorsal skin (153) Delayed-type contact hypersensitivity (DTH)(92, 154) Wound Repair (155) Gut Intestinal mucosal injury (156, 157)Inflammatory injury (158) Early phase histamine-induced inflammation(159) Ischemia-reperfusion injury (160) Inflamed peritoneum (94)Intestinal ischemia-reperfusion injury (161) Inflammatory bowel disease(162) Acute injury to peritoneum (163) Abdominal inflammation (164)Colon carcinoma (95, 165) Intestinal inflammation (166) KidneyNeutrophil-dependent glomerular injury (167) Acute passiveantiglomerular basement membrane (114) nephritis Glomerulonephritis(168) Ischemia-reperfusion injury (169, 170) CirculatoryInflamed/injured venular endothelium (171) Splanchnicischemia-reperfusion injury (172, 173) Myocardial ischemia-reperfusioninjury (100-102, 105, 174, 174- 178, 178, 179) PMN leukocyte-inducedvasocontraction and (107) endothelial dysfunctions Severe vasculartrauma (180) Atherogenesis brought on by oxidized LDL (181) Inflammationand thrombosis (182) Hemorrhagic shock injury (183) Reduced thrombisformation (184, 185) Atherosclerotic vascular disease (186) Disruptedblood hemostasis (187) Increased infection susceptibility (143) Alteredhematopoiesis (143) Grey platelet syndrome (188) Peripherial arterialdisease (109) Endothelial cell hypoxia and hypoxia regeneration (189,190) Enhanced heart allograft afteriosclerosis (191)Hypercholesterolemia (192) Angina pectoris/Unstable angina (193, 194)Coronary spasm (195) Vasocontraction and endothelial cell dysfunction(107) Atherogenesis and hypertension (186) CNS Ischemia-reperfusioninjury (115) Brain meningitis (196) Eye Intraocular inflammation (197)Misc. Hypertension of the adrenal glands (198) Increased expression ofP-selectin in breast cancer (199) tissue Hyperthyroidism (121) Tumorangiogenesis (200)

J. Pediatr. Surg. 32:1010-1013 (1997); Davenpeck et al., Am. J. Physiol.Heart Circ. Physiol. 267:H622-H630 (1994); Gauthier et al., Am. J.Physiol. Gastrointest. Liver Physiol. 267:G562-G568 (1994); Weyrich etal., J. Clin. Invest. 91:2620-2629 (1993); Tojo et al., Glycobiology6:463-469 (1996); Lefer et al., Am. J. Physiol. Heart Circ. Physiol.271:H2421-H2429 (1996)). Thus, such disorders can be treated using amethod of the invention such that the severity of the pathology isreduced. Similarly, acute and chronic inflammatory disorders can betreated using a method of the invention.

For the methods of the invention, an amount of heparin that does notproduce substantial anticoagulant activity or undesirable bleeding isadministered to the subject. As used herein, reference to “an amount ofheparin that does not produce substantial anticoagulant activity” meansan amount of heparin that does not cause bleeding complications,although a mild anticoagulant effect can occur. Thus, the amount ofheparin administered generally will result in a plasma heparin level ofless than about 0.2-0.4 units/ml plasma in a subject.

Clinical signs and symptoms of undesirable bleeding includes blood inthe urine, or stool, heavier than normal menses, nose bleeds orexcessive bleeding from minor wounds or surgical sites. Easy bruisingcan precede such clinical manifestations. Where undesirable bleedingoccurs, heparin activity can be neutralized by administration ofprotamine sulfate.

Although an amount of heparin administered to inhibit L-selectin andP-selectin mediated binding in a subject will depend, in part, on theindividual, normal adult subjects administered heparin in amounts thatresult in less than 0.2 units heparin/ml of plasma generally do notexhibit undesirable bleeding. A subject treated with heparin can bemonitored for undesirable bleeding using various assays well known inthe art. For example, blood clotting time, active partial thromboplastintime (APTT), or anti-Xa activity can be used to determine if coagulationstatus is undesirably increased in a subject administered heparin. Whereundesirable bleeding occurs, heparin administration is discontinued.

The amount of heparin administered depends, in part, on whetherL-selectin or P-selectin mediates binding and, therefore, whether onlyP-selectin, or both L-selectin and P-selectin, are to be inhibited. Forexample, an amount of heparin less than that used for anticoagulanttherapy can be administered to a subject for the purpose ofsubstantially inhibiting P-selectin as compared to L-selectin (see Table5 and FIGS. 8 and 9). The amount of heparin administered to a subjectalso depends on the magnitude of the therapeutic effect desired.

In addition, the amount of heparin administered will depend on theindividual subject because the bioavailability of heparin withinsubjects is known to vary. For example, heparin dosages are sometimesadministered in units heparin/kg body weight. However, the dosages ofheparin needed (e.g. units heparin/kg body weight) to attain specificlevels of heparin in the plasma of a subject can vary among individualsbecause of differences in heparin bioavailability. Thus, the heparinconcentration in the blood of a subject in units/ml plasma is the morereliable measure of heparin concentration. The amount of plasma heparinin a subject can be determined using titration and neutralization assayswith protamine sulfate.

Heparan sulfate (HS) chains released from endothelial cell heparansulfate proteoglycans (HSPGs) bind to L-selectin (Norgard-Sumnicht andVarki, J. Biol. Chem. 270:12012-12024 (1995)). Selectin-Receptorglobulin (Rg) chimeric proteins were used to make affinity columns todetermine if these HS ligands also bound to other selectins (P and E).Metabolically labeled HS chains obtained from two endothelial cell lineswere applied to each selectin affinity column (see Example I). HS ligandbound primarily to the L-selectin and P-selectin column, but not to theE-selectin column (see FIG. 1). The HS ligand elution profiles using 5mM EDTA buffer were different; all ligand was released from L-selectin,whereas there was poor release of ligand from P-selectin (see FIG. 1C,inset). Thus, a P-selectin affinity column completely bound theL-selectin HS ligands from both endothelial cell types, but showed acomparatively slow and partial elution with 5 mM EDTA. Addition of 20 mMEDTA resulted in recovery of all of the labeled ligand from both cellsources. This difference in HS elution between the L-selectin andP-selectin columns indicates that the eluting effect of EDTA withP-selectin might not be strictly based upon its calcium chelatingproperties (see below).

High affinity interactions of selectin generally are dependent oncalcium. In addition to calcium, magnesium can potentiate the effects ofcalcium in P-selectin binding (Geng et al., Nature 343:757-760 (1990)).However, the HS ligands studied here are structurally different from thepreviously described sialylated ligands (Varki, Proc. Natl. Acad. Sci.USA 91:7390-7397 (1994); Furie and Furie, Thromb. Haemost. 74:224-227(1995); Vestweber, J. Clin. Invest. 98:1699-1702 (1996); McEver et al.,J. Biol. Chem. 270:11025-11028 (1995); Nelson et al, Annu. Rev. CellBiol. 11:601-631 (1995); Springer, Annu. Rev. Physiol. 57:827-872(1995); Crocker and Feizi, Curr. Opin. Struct. Biol. 6:679-691 (1996);Kansas, Blood 88:3259-3287 (1996); Rosen and Bertozzi, Curr. Biol.6:261-264 (1996); Vestweber, J. Cell. Biochem. 61:585-591 (1996); Varki,J. Clin. Invest. 99:158-162 (1997); Phillips et al., Science250:1130-1132 (1990); Walz et al., Science 250:1132-1135 (1990); Polleyet al., Proc. Natl. Acad. Sci. USA 88:6224-6228 (1991); Berg et al., J.Biol. Chem. 266:14869-14872 (1991); Tyrell et al., Proc. Natl. Acad.Sci. USA 88:10372-10376 (1991); Berg et al., Biochem. Biophys. Res.Commun. 184:1048-1055 (1992); Foxall et al., J. Cell Biol. 117:895-902(1992); Larkin et al., J. Biol. Chem. 267:13661-13668 (1992); Rosen andBertozzi, Curr. Opin. Cell Biol. 6:663-673 (1994)) and, therefore, ananalysis of the calcium dependence of heparan sulfate chain binding toL-selectin and P-selectin was performed.

The calcium dependence of L-selectin binding was determined usingbinding buffer without added calcium. The binding was not changed,although trace amounts of calcium (approximately 50 μM) present in amagnesium-containing buffer may support L-selectin binding. Indeed,binding of HS to L-selectin was abolished in magnesium/EGTA buffers, inwhich calcium was lowered to negligible amounts (FIG. 2A). In contrast,binding of the HS chains to P-selectin occurred in the magnesium/EGTAbuffer (FIG. 2B), indicating that HS binds to P-selectin in the absenceof exogenously added calcium. FIG. 3 further demonstrates that bindingby P-selectin in the absence of exogenously added cations still requires20 mM EDTA buffer for elution of the HS chains. These results show thatalthough the binding specificities of L-selectin and P-selectin overlap,there are differences, including the different requirement for calcium.However, the presence of calcium in vivo indicates that heparin willinhibit the binding of L-selectin in a subject.

The need for 20 mM EDTA to elute HS chains from the P-selectin column inthe absence of divalent cations indicates that either trace amounts ofcalcium remain tightly bound to the selectin column from the priorexperiments or that EDTA is eluting the ligand from P-selectin by amechanism unrelated to its divalent cation chelating properties.However, it is unlikely that trace amounts of calcium remain tightlybound to the selectin column because crystal structures of E-selectinand the related C-type lectin mannose binding protein indicate that thecalcium ion is exposed to the solvent and does not lie deeply buried inthe binding pocket (Graves et al., Nature 367:532-538 (1994); Weis etal., Nature 360:127-134 (1992)). More likely, EDTA is eluting the ligandfrom P-selectin by a mechanism unrelated to its divalent cationchelating properties because an equivalent (80 mM) increase in chlorideconcentration did not cause elution. Thus, EDTA can elute the HS ligandsdue to its inherent polycarboxylate structure, for example, by mimickingthe high charge density of HS chains.

HS chains are available in limited quantities so the ability ofcommercially available mast cell derived PIM-heparin to bind to theselectins was examined (Example II). In addition, PIM-heparin is used asan anticoagulant in humans and its potential use as a selectintherapeutic was of interest. Labeled PIM-heparin in a column bufferadjusted to more closely approximate physiological conditions (Koenig etal., Glycobiology 7:79-93 (1997)) was applied to each of the threeselectin affinity columns and the bound material was eluted (FIG. 4).

Similar to the results obtained for HS chains, none of the PIM-heparinmolecules bound to E-selectin (FIG. 4A). 90% of the total PIM-heparinloaded bound to L-selectin; 58% was eluted with 2 mM EDTA and 32% waseluted with 20 mM EDTA (FIG. 4B). In contrast, 79% of the PIM-heparinbound to P-selectin, very little was eluted with 2 mM EDTA but 20mM EDTAeluted all of the bound heparins (FIG. 4C). These results demonstratethat the binding of PIM-heparin to the selectins was similar to the HSchains obtained from endothelial cells, except that a portion ofPIM-heparin remained bound to L-selectin after adding 2 mM EDTA. Thus,heparin compositions commonly formulated for use as anticoagulants havethe ability to bind to L-selectin and P-selectin.

The calcium dependence of PIM-heparin binding to L-selectin andP-selectin was similar to the binding of the endothelial HS chains tothe selectins (Example II and FIG. 5). Binding to L-selectin wasabolished in magnesium/EGTA buffers (FIG. 5A), while a significantfraction continued to bind to P-selectin under these conditions (FIG.5B). However, the fraction that bound to P-selectin was lower than thatseen under calcium-replete conditions (45% vs. 79%), possibly indicatinga partial calcium dependency of the initial binding event. Of thefraction that bound in the absence of calcium, 20 mM EDTA was requiredfor elution (FIG. 5B). These results indicate that calcium occupancy inthe lectin site may assist the initial binding of some heparin fragmentsto P-selectin. However, once bound, calcium does not appear to berequired to maintain the interaction between the heparin chains andP-selectin.

PIM-heparin chains have a large molecular weight (average 20 kDa;approximately 38-40 disaccharide units) and are polydisperse instructure (Pervin et al., Glycobiology 5:83-95 (1995)). In order todetermine the specificity of interaction between L-selectin and heparin,the binding of size-fractionated heparin fragments to an L-selectincolumn was examined (Example III). The binding of PIM-heparin fragmentsto L-selectin and P-selectin was size dependent; substantial bindingoccurred with tetradecasaccharides (see FIG. 6D and Table 2). Theseresults indicate that the fraction of PIM-heparin that binds toL-selectin and P-selectin predominantly comprises tetradecasaccharidesor larger fragments.

Heparin tetrasaccharides interact with L-selectin in blocking binding toimmobilized sialyl-Lewis^(x) (SLe^(x); Siao2-3 Galβ1-4 (Fucal-3)GlcNAc), a well known component of natural selectin ligands (Nelson etal., supra, 1993). Studies of other oligosaccharide protein interactionsindicate that binding constants in the low μM range would be needed forthe detection of selectin binding (Varki and Kornfeld, J. Biol. Chem.

TABLE 2 Unfractionated and LMW Heparins as Inhibitors of L- andP-Selectin binding to Immobilized SLc^(x) and to [³H] HL-60 cells.Therapeutic IC₅₀ Units/ml* range Against immobilized SLc^(x) AgainstHL-60 cells Clinical heparin (Units/ml)* E-Selectin L-SelectinP-Selectin L-Selectin P-Selectin Unfractionated Heparin 0.2-0.4 >500.07-0.08 0.01-0.02 0.02-0.03 0.003-0.01  Levonox (LMW Heparin,Enoxaparin) 0.6-1.0 >50 0.8-1.5 0.8-1.0 1.5-3.0 0.7 Fragmin (LMWHeparin, Deltaparin) 0.6-1.0 >50 0.7-2.0 1.5-2.0 4.0-7.0 1.0-4.0

Several clinical lots of Unfractionated and LMW Heparins were tested fortheir ability to inhibit selectin binding to immobilized Sle^(x) inELISA inhibition experiments or HL-60 cells binding to immobilizedselectins, as described under “Materials and Methods.” IC₅₀ values weredetermined by the equation: {[(average of duplicates)−(average ofnegative controls)]/[(average of positive controls)−(average of negativecontrols)]}×100, where the positive controls were without inhibitors,and the negative controls contained 5 mM EDTA in the ELISA inhibitionexperiments, and 20 mM EDTA in the HL-60 cell inhibition experiments.Experiments were performed 2-times, and the range of measured IC₅₀values are presented. Examples of the results can be seen in FIGS. 1 and2. Unfractionated heparin concentrations are reported in Protamineneutralization Units while LMW heparins are reported as anti-Xa Units(see text for discussion). 258:2808-2818 (1983); Powell et al., J. Biol.Chem. 270:7523-7532 (1995)). ELISA inhibition experiments usingimmobilized SLe^(x) demonstrated that the PIM-heparintetradecasaccharides had a calculated IC₅₀ value of 82 μM and 54 μM forL-selectin and P-selectin binding, respectively (see Example IV andTable 3). These IC₅₀ values are almost 10-fold less than those reportedfor Sle^(x) itself (520 μM and 600 μM for P-selectin and L-selectin,respectively). These results demonstrate that the tetradecasaccharidesbind L-selectin and P-selectin with greater affinity than SLe^(x).

Given the size of the tetradecasaccharides relative to that of theselectin lectin domain, the interactions with tetradecasaccharideslikely represent monovalent recognition by the selectin-Rg chimeras(Graves et al., supra, 1994). The tetradecasaccharides were generated bypartial heparin lyase I degradation and reduced with borohydride whichresults in the first, or nonreducing, monosaccharide unit having anon-native C4-C5 double bond, and the last unit having an open ring.Consistent with the results discussed above, the binding to L-selectinwas disrupted by 2 mM EDTA and was calcium dependent whereas the bindingto P-selectin was calcium independent. The crystal structure of theclosely related molecule E-selectin indicated that the putativecarbohydrate binding region of the lectin domain is located on theopposite face from the EGF domain. Thus, despite the lack of calciumdependence of binding of the tetradecasaccharides to P-selectin, thesemolecules may be binding somewhere relatively close to the calciumdependent binding site for SLe^(x).

TABLE 3 Heparin Fragment Mixtures as Inhibitors of L- and P-Selectinbinding to Immobilized SLc^(x). IC₅₀ value L-Selectin P-SelectinTetradecasaccharides   54 uM   82 uM Dodecasaccharides  159 uM  159 uMDecasaccharides >1900 uM >1900 uM Octasaccharides >2400 uM >2400 uMSialyl Lewis^(x)  600 uM  520 uM

Size-fractionated PIM-heparin mixtures were tested for their ability toinhibit selectin binding to immobilized SLe^(x) in ELISA inhibitionexperiments, as described under “Materials and Methods.” There was noinhibition of E-selectin, even with the tetradecasaccharides, atconcentrations as high as 5 mg/ml (>1000 uM). IC₅₀ values for L- andP-selectin inhibition were determined by the equation: {[(average ofduplicates)−(average of negative controls)]/[(average of positivecontrols)−(average of negative controls)]}*100, where the positivecontrols were without inhibitors, and the negative controls contained 5mM EDTA. Experiments were performed 2-3 times, and the averaged IC₅₀values are presented. For calculation or concentration, the followingaverage molecular weights were assumed: tetradecasaccharide, 3675 Da;dodecasaccharide, 3150 Da; decasaccharide, 2625 Da; and octasaccharides,2100 Da. The inhibitory potency of the tetrasaccharide Sialyl Lewis^(x)under the same conditions is shown for comparison.

With the availability of a size defined fraction, the binding of somemolecules and the lack of binding of others indicates a degree ofspecificity. Such specificity was confirmed by performing rebindingexperiments with the tetradecasaccharide pooled fractions were performed(Example III). For both L-selectin and P-selectin, the binding of thefractions was reproducible (see FIG. 7, for L-selectin, and Table 4).The divalent cation dependence of this interaction also was similar tothat of intact heparin and the endothelial HS chains. In addition, theinteractions with L-selectin were disrupted by 2 mM EDTA (FIG. 7),whereas those with P-selectin required 20 mM EDTA for disruption(Table4) and studies with magnesium/EGTA buffers confirmed the calciumdependency of L-selectin binding. These results demonstrate that variousligand populations differ over a wide range in their ability to interactwith L-selectin. Thus, individual heparin chains or pools of relatedchains are likely to exhibit differential affinity for selecting. Suchligands that selectively bind are useful for the selective targeting ofselectins in vivo.

In an effort to determine if there are differences between the types oftetradecasaccharides that are recognized by L-selectin and P-selectin,cross-binding studies were performed (Example III). Of thetetradecasaccharides that bound to L-selectin, 100% bound to P-selectinand were eluted with 20 mM EDTA (see Table 4). In contrast, not all ofthe tetradecasaccharides that bound to P-selectin also bound toL-selectin. Of the tetradecasaccharides from the P-selectin column thatbound to L-selectin, 22% were from the 2 mM EBTA eluted fraction and 51%were from the 20 mM DTA eluted fraction. Of the fraction that did notbind

TABLE 4 Rebinding and cross-binding of Tetradecasaccharides to L- andP-selectin Original Binding of sample L-selectin P-selectin P-selectinL-selectin 2 mM EDTA P-selectin 2 mM EDTA 20 mM EDTA Secondary Bindingunbound eluted unbound eluted eluted L-selectin Unbound 98%  0% 100% 78% 49% 2 mM EDTA eluted  2% 100%  0% 22% 51% P-selectin Unbound 88%  0%88%  0%  0% 2 mM EDTA eluted  0%  0%  5% 100%   0% 20 mM EDTA Eluted 12%100%  7%  0% 100% 

[³H]-labeled tetradeccasaccharides were fractionationed by P- andL-selectin affinity chromatography as described under “Materials andMethods”). After desalting, aliquots of each fraction were reapplied tothe same column, or to the other selectin column, (see FIG. 7 for anexample of the results). to L-selectin, 12% re-bound to P-selectin.These data indicate that, although both L-selectin and P-selectinexhibit some degree of selectivity, L-selectin has a greater degree ofselectivity than P-selectin in binding heparin tetradecasaccharides.Thus, in view of this greater selectivity, L-selectin can be targetedover P-selectin using particular tetradecasaccharide fractions.

Since the size of the recognized tetradecasaccharides is not a variable,the differences in affinity are a result of structural differencesbetween them. The structures of the tetradecasaccharides were examinedusing enzymes or chemical modification that cleave the chains atparticular sites (Lohse and Linhardt, J. Biol. Chem. 267:24347-24355(1992); Conrad, In Current Protocols in Molecular Biology 17-22A (1996);Desai et al., Biochemistry 32:8140-8145 (1993); Jandik et al.,Glycobiology 4:289-296 (1994); Linhardt, In Current Protocols inMolecular Biology 17-18B (1996)). Bound and flow-through material fromthe P-selectin and L-selectin columns were cleaved, and the productsanalyzed by FPLC (Example III). L-selectin binding fragments were moreheavily sulfated and epimerized and, similar to the endothelial HSchains, they were enriched in free amino groups. The P-selectin bindingcomponent included this fraction as well as some less highly modifiedregions; they were sensitive to Heparin Lyase III. These resultsindicate that, while distinct structural features selectively enhanceinteractions of P-selectin and L-selectin with heparintetradecasaccharides, the binding represents a continuum of affinitieswith no apparent structural motif being markedly superior in its abilityto be recognized.

The clinical relevance of the observations made using commerciallyavailable PIM-heparin, which is very similar to the heparin used as ananticoagulant (Ginsberg, N. Engl. J. Med. 335:1816-1828 (1996); Hirsh,Semin. Thromb. Hemost. 22 Suppl:7-12 (1996); Pineo and Hull, Annu. Rev.Med. 48:79-91 (1997)) were analyzed further. ELISA inhibition studiesusing immobilized SLe^(x) indicated that crude commercial PIM-heparinhad an IC₅₀ of 18 μg/ml and 2 μg/ml for SLe^(x) binding to L-selectinand P-selectin, respectively. Although crude heparin preparations varybetween lots, the IC₅₀ values for crude PIM-heparin roughly correspondsto pharmaceutical heparin preparations of approximately 0.18-0.09units/ml (L-selectin) and 0.02-0.01 units/ml (P-selectin). These resultsindicate that the inhibition of binding can occur at therapeuticallyrelevant concentrations.

In order to understand the relevance of these studies to clinicalpractice, the effect of pharmaceutical heparin preparations on selectinbinding was determined (Example IV). ELISA inhibition assays (see FIG. 8and Table 5) demonstrated that two separate lots of pharmaceuticalunfractionated heparin significantly inhibited L-selectin andP-selectin, but not E-selectin, binding at concentrations less than thatrecommended for anticoagulation therapy (Table 5). The calculated IC₅₀values towards SLe^(x) binding were 0.07-0.08 units/ml for L-selectinand 0.01-0.02 units/ml for P-selectin (Table 5).

Similar findings were obtained in an HL-60 cell attachment assay, whichmeasured the interaction of L-selectin and P-selectin with the naturalselectin ligand PSGL-1 expressed by the HL-60 cells (Example IV). PSGL-1is a primary selectin ligand with significant

TABLE 5 Unfractionated and LMW Heparins as Inhibitors of L- andP-Selectin binding to Immobilized SLc^(x) and to [³H] HL-60 cells.Therapeutic IC₅₀ Units/ml* range Against immobilized SLc^(x) AgainstHL-60 cells Clinical heparin (Units/ml)* E-Selectin L-SelectinP-Selectin L-Selectin P-Selectin Unfractionated Heparin 0.2-0.4 >500.07-0.08 0.01-0.02 0.02-0.03 0.003-0.01  Levonox (LMW Heparin,Enoxaparin) 0.6-1.0 >50 0.8-1.5 0.8-1.0 1.5-3.0 0.7 Fragmin (LMWHeparin, Deltaparin) 0.6-1.0 >50 0.7-2.0 1.5-2.0 4.0-7.0 1.0-4.0

Several clinical lots of Unfractionated and LMW Heparins were tested fortheir ability to inhibit selectin binding to immobilized SLe^(x) inELISA inhibition experiments or HL-60 cells binding to immobilizedselectins, as described under “Materials and Methods.” IC₅₀ values weredetermined by the equation: {[(average of duplicates)−(average ofnegative controls)]/[(average of positive controls)−(average of negativecontrols)]}×100, where the positive controls were without inhibitors,and the negative controls contained 5 mM EDTA in the ELISA inhibitionexperiments, and 20 mM EDTA in the HL-60 cell inhibition experiments.Experiments were performed 2-3 times, and the range of measured IC₅₀values are presented. Examples of the results can be seen in FIGS. 8 and9. Unfractionated heparin concentrations are reported in Protamineneutralization Units while LMW heparins are reported as anti-Xa Units(see text for discussion). biological relevance in vivo (Kansas, supra,1996; Rosen and Bertozzi, supra, 1996; Vestweber, supra, 1996; Lowe andWard, J. Clin. Invest. 99:822-826 (1997); Varki, supra, 1997; McEver andCummings, J. Clin. Invest. 100:485-492 (1997)). The binding of HL-60cells to L-selectin and to P-selectin was inhibited by unfractionatedpharmaceutical heparin (FIG. 9). The IC₅ values calculated for HL-60binding to L-selectin and P-selectin were approximately 12-fold and50-fold lower, respectively, than the recommended range foranticoagulant therapy (Table 5).

Pharmaceutical preparations of low molecular weight (LMW) heparinsdifferent from those described by Bevilacqua et al. have been used foranticoagulant therapy in place of unfractionated heparin due to enhancedbioavailability and half-life and a somewhat decreased degree oftoxicity (Ginsberg, supra, 1996; Hirsch, supra, 1996; Sakuragawa andTakahashi, Semin. Thromb. Hemost. 16 Suppl.:5-11 (1990); Pineo and Hull,supra, 1997; Boneu, Semin. Thromb. Hemost. 22:209-212 (1996)). Theability of two kinds of clinical grade LMW heparins, FRAGMIN(Pharmacia-Upjohn; Kalamazoo Mich.) and LOVENOX (Rhone-Poulenc Rorer;Collegeville Pa.), to inhibit selectin binding was compared tounfractionated pharmaceutical heparin (FIG. 8). The IC₅₀ values of theLMW heparins for L-selectin and P-selectin binding were at or higherthan the recommended therapeutic levels of unfractionated heparin inELISA inhibition experiments against immobilized SLe^(x) and in HL-60cell attachment inhibition experiments (Table 5). Thus, the two LMWheparins are much poorer inhibitors of L-selectin and P-selectin bindingto sialylated ligands, including the naturally occurring PSGL-1 ligand,than unfractionated pharmaceutical heparin. The difference in bindinginhibition between unfractionated and LMW heparins appears to be due tosmaller sized heparin fragments having less affinity or avidity for theselectins (see FIG. 6).

The results disclosed herein demonstrate that L-selectin and P-selectinbinding to SLe^(x) and to PSGL-1 of HL-60 cells is inhibited byunfractionated pharmaceutical heparin preparations at concentrations12-fold to 50-fold lower than those recommended for effectiveanticoagulation in vivo. These results indicate that patients undergoingheparin anticoagulant therapy can experience clinically significantinhibition of L-selectin and P-selectin function. However, the currentswitch from unfractionated heparin to low molecular weight anticoagulantforms of heparin likely results in a significant loss of the selectininhibitory effect due to the decreased ability of LMW heparins toinhibit L-selectin and P-selectin binding. This relative lack ofaffinity may be due to selectins favoring higher order carbohydratestructures. As disclosed herein, administration of low doseunfractionated heparin provides a treatment option for acute and chronicdiseases involving P-selectin or L-selectin mediated binding,particularly administration of heparin in an amount that does notproduce substantial anticoagulant activity or undesirable bleeding in asubject.

Although SLe^(x) and related structures may be therapeutically useful ina variety of disease states (Albelda et al., FASEB J. 8:504-512 (1994);Lowe and Ward, supra, 1997; Weyrich et al., J. Clin. Invest.91:2620-2629 (1993); Ridings et al., J. Appl. Physiol. 82:644-651(1997); Bevilacqua et al., Annu. Rev. Med. 45:361-378 (1994); Skurk etal., Am. J. Physiol. Heart Circ. Physiol. 267:H2124-H2131 (1994); Lefer,Ann. Thorac. Surg. 60:773-777 (1995); Seekamp and Ward, Agents Actions41:137-152 (1993)), they have a number of significant drawbacks. Asdisclosed herein, the affinity of pharmaceutical compositions ofunfractionated heparin for L-selectin and P-selectin is higher than thatof oligosaccharides such as SLe^(x) (Table 3), LOVENOX and FRAGMIN(Table 5 and FIG. 8). In particular, the IC₅₀ calculated for inhibitionof HL-60 cell attachment to L-selectin using unfractionated heparin isapproximately 10-fold less than the recommended levels foranticoagulation (Table 5). The IC₅₀ values for P-selectin are evenlower, and are approximately 50-fold less than the recommendedtherapeutic level for anticoagulant activity. Thus, the recommendedlevel of unfractionated heparin for anticoagulation can completelyinhibit L-selectin and P-selectin mediated binding in an in vitrobinding assay.

In addition to FRAGMIN and LOVENOX, other forms of LMW heparins used foranticoagulant therapy are commercially available. As disclosed herein,these other LMW heparins also can be useful for inhibiting selectinbinding at concentrations less than those used for anticoagulanttherapies. Such forms of LMW heparin formulated for clinical use inanticoagulant therapies, therefore, also can be used in a method of theinvention.

Many reperfusion injury situations such as those occurring in stroke andmyocardial ischemia, in which inhibition of PSGL-1 based P-selectin andL-selectin interactions are a potential target (Table 1), are routinelytreated with anticoagulating amounts of heparin with the intention ofpreventing further thrombosis. As disclosed herein, a result ofadministering these amounts of heparin is that clinicians may haveinadvertently inhibited selectin mediated binding. Consistent with thisview, the best effects of thrombolytic therapy for acute myocardialinfarction occurs when heparin is included in the treatment regimen. Theresults disclosed herein indicate that heparin can provide protectionfrom reperfusion injury by inhibiting P-selectin and L-selectin mediatedbinding.

The results disclosed herein, along with the established record ofheparin as a therapeutic agent, indicate that heparin can be useful forinhibiting L-selectin or P-selectin based interactions using amountslower than those required for anticoagulant therapy. Thus, the inventionprovides a method of inhibiting L-selectin or P-selectin binding in asubject, by administering to the subject an amount of heparin that doesnot produce substantial anticoagulant activity or undesirable bleedingin the subject. Further provided are methods of treating an L-selectinor P-selectin related pathology by administering to a subject having thepathology an amount of heparin that does not produce substantialanticoagulant activity or undesirable bleeding in the subject.

Particular acute and chronic conditions, in which P-selectin orL-selectin have a pathophysiological role can be treated using a methodof the invention (Table 1). For example, undesirable immune responses inwhich the homing or adhesion of leukocytes, neutrophils, macrophages,eosinophils or other immune cells mediated by the interaction ofL-selectin with endothelial cell ligands, can be inhibited byadministering heparin to the subject according to a method of theinvention. Inhibition of neutrophil adherence, for example, caninterrupt the cascade of damage initiated by free oxygen radicalsecretion and related activities that result in tissue damage and lossof myocardial contractile function present in myocardial infarction.Similarly, P-selectin mediated adhesion of cells such as neutrophils andplatelets can be inhibited in a subject if this activity is undesirable.Thus, the severity of chronic immune disorders or acute inflammatoryresponses can be reduced using a method of the invention.

When administered to a subject, heparin is administered as apharmaceutical composition. Such pharmaceutical compositions of heparinare commercially available and protocols for heparin administration arewellknown in the art. Such compositions and administration protocols canbe conveniently employed in practicing the invention. One skilled in theart would know that the choice of the particular heparin pharmaceuticalcomposition, depends, for example, on the route of administration andthat a pharmaceutical composition of heparin can be administered to asubject by various routes., including, for example, parenterally,particularly intravenously. The heparin composition can be administeredby intravenous or subcutaneous injection, and administration can be as abolus or by continuous infusion. In addition, mucosally absorbable formsof heparin can be administered orally, rectally or by inhalation,provided the amount of heparin attained in the blood does not exceed aconcentration of about 0.2-0.4 units/ml plasma and does not producesubstantial anticoagulant activity or undesirable bleeding in thesubject.

Depending on the commercial source, pharmaceutical preparations ofheparin for injection are supplied at concentrations from 1000 units/mlto 20,000 units/ml (Eli Lilly, Indianapolis Ind.; Elkins-Sinn, Inc.,Cherry Hill N.J.; Wyeth-Ayerst Laboratories, Philadelphia Pa.;Pharmacia-Upjohn, Kalamazoo Mich.). Thus, pharmaceutical heparinpreparations that are about one-fifth to one-tenth as concentrated canbe used for administration to a subject in order to inhibit L-selectinor P-selectin mediated binding in the subject. For example, heparinpharmaceutical compositions of about 250-950 units/ml or 50-250units/ml, or less can be prepared and used in the methods of theinvention. Thus, the invention provides pharmaceutical compositionscomprising less than 1000 units heparin/ml. Dilutions of moreconcentrated commercially available pharmaceutical heparin to achievethese lower concentrations of heparin also are provided.

The following examples are intended to illustrate but not limit thepresent invention.

EXAMPLE I Binding of Heparan Sulfate Chains to L-Selectin and P-Selectin

This example demonstrates that heparan sulfate (HS) chains fromendothelial cells bind to L-selectin and P-selectin.

A. Endothelial Heparan Sulfate Ligands for L-selectin also Bind toP-selectin but not to E-selectin

Affinity columns containing the L-selectin, P-selectin and E-selectinchimeric proteins were used to determine whether HS ligands releasedfrom endothelial cell HSPGs that bind to L-selectin, also can bind otherselectins. Recombinant selectin chimeras consisting of L-selectin-Rg,P-selectin-Rg and E-selectin-Rg were produced as previously described(Norgard et al., Proc. Natl. Acad. Sci. USA 90:1068-1072 (1993); Aruffoet al., Proc. Natl. Acad. Sci. USA 89:2292-2296 (1992); Nelson et al.,J. Clin. Invest. 91:1157-1166 (1993)). Affinity columns were prepared byimmobilizing 0.5 mg of each selectin-Rg on 0.5 ml of protein A-SEPHAROSE(PAS; Sigma, St. Louis Mo.).

CPAE cells, a calf pulmonary artery endothelial cell line (ATCCAccession No. CCL 209), were used at or before passage 23 as a source ofHS chains and were grown to 80% confluence. Human umbilical veinendothelial cells (HUVECs; Clonetics, San Diego Calif.) were used withinthe first 3 passages and also provided a source of HS chains. CPAE andHUVECcells were labeled with (³H)GlcNH₂ (60 Ci/mmol; AmericanRadiolabeled Chemicals, St. Louis Mo.) and the free HS chains werereleased from the (³H)GlcNH₂ labeled cell proteoglycans and purified asdescribed previously (Norgard-Sumnicht et al., supra, 1993;Norgard-Sumnicht and Varki, supra, 1995; Roux et al., J. Biol. Chem.263:8879-8889 (1988)). Similar amounts of the labeled HS chains in 100mM NaCl, 20 mM MOPS (pH 7.4), 1 mM CaCl₂, 1 mM MgCl₂ buffer were appliedto each column. After washing the column, bound ligand was eluted usingthe above binding buffer, except CaCl₂ and MgCl₂ were replaced with 5 mMEDTA.

Most of the HS ligand rebound to the L-selectin column and was elutedwith 5 mM EDTA (FIG. 1A). The HS ligand also bound to the P-selectincolumn, however, it eluted very poorly with 5 mM EDTA (FIG. 1C, inset).Subsequent elution with binding buffer in which CaCl₂ and MgCl₂ wasreplaced with 20 mM EDTA resulted in recovery of all of the HS ligandfrom the P-selectin column. In contrast, no binding to the E-selectincolumn was detected (FIG. 1B), although the E-selectin column did bindother ligands from carcinoma cells in a calcium-dependent manner and,therefore, was functional. (³⁵S)-labeled HS chains from CPAE cells andlabeled HS chains from HUVEC cells provided similar binding profiles tothe L-selectin and P-selectin columns.

These results demonstrate that HS ligands obtained from CPAE and theHUVEC cells bound the L-selectin and P-selectin columns, but notE-selectin column.

B. Comparison of Calcium Dependence of Heparan Sulfate Chain Binding toL-selectin and P-selectin

The calcium dependence of the HS chain interaction with L-selectin andP-selectin was determined. Aliquots of HS chains that previously boundto an L-selectin column were reapplied either to the same column or to aP-selectin column. The columns were washed, eluted with either 5 mM EDTAfor L-selectin or 20 mM EDTA for P-selectin, and the fractions collectedand monitored for radioactivity (FIG. 2).

Omission of calcium from the binding buffer did not change the results,although calcium (approximately 50 μM) present in the 2 mM magnesiumcontaining buffer may support L-selectin binding. The binding of HSchains to L-selectin was abolished in Mg⁺⁺/EGTA buffers, in whichcalcium is present in negligible amounts (effective Ca⁺⁺ concentration0.774 nM, Mg⁺⁺ concentration 3 mM; FIG. 2). The calcium dependence ofthe L-selectin interaction was confirmed by demonstrating that 0.5 mMEDTA efficiently eluted HS chains from the L-selectin column (FIG. 2A).In contrast to L-selectin, binding of the HS chains to P-selectinoccurred even in the magnesium/EGTA buffer (see FIG. 2B, indicating thatHS binding to P-selectin did not require exogenously added calcium.

Since the binding of the HS chains to P-selectin occurred in thepresence of either calcium or magnesium, the binding requirements forexogenously added cations was determined. An aliquot of the HS-GAGligand that had previously bound to an L-selectin column was dialyzedagainst water, then was adjusted into buffer containing 100 mM NaCl, 20mM MOPS (pH 7.4). The P-selectin column was prepared by washing with 20mM EDTA, incubating in 20 mM EDTA overnight at 4° C., then extensivelywashing with the MOPS/NaCl buffer. The HS-GAG aliquot was applied to theP-selectin column, washed with the same buffer and eluted with 20 mMEDTA. The fractions were collected and monitored for radioactivity.

Binding to P-selectin occurred in the absence of exogenously addedcations (FIG. 3); however, the ligand was eluted with 20 mM EDTA. Theseresults show that L-selectin binding is divalent cation dependent,whereas P-selectin binding does not require exogenously added divalentcations.

EXAMPLE II Binding of Porcine Mucosal Heparin to L-Selectin andP-Selectin

This example demonstrates that porcine intestinal mucosal (mastcell-derived) heparin (PIM-heparin) binds to L-selectin and P-selectin.

A. PIM-heparin Shows Similar, but not Identical, Binding to theSelecting

The binding of commercially available PIM-heparin (Sigma, St. Louis Mo.)to L-selectin and P-selectin was analyzed. Briefly, the large heparin,chains in this preparation were radiolabeled by adding a 5-fold molarexcess of NaB(³H)₄ (approximately 1.4 to 7 mCi) to about 20 to 120nmoles of di- to tetradeca-saccharide heparin in 100 μl of 0.2 M sodiumborate buffer, pH 10. The reaction proceeded for 4 hours at roomtemperature and 40 μl of 1 M NaBH₄ was added. After one hour, 40 μl ofacetone was added to quench the reaction. The next day, the samples weretreated with 150 μl of 1.0 M ammonium formate buffer and stored at −80°C. until use.

Thawed samples were desalted using Bio-Gel P-2 columns (Bio-Rad,Hercules Calif.) in 100 nM ammonium formate buffer, pH 5. The voidvolume (Vo) and included volume (Vi) were determined by loading 400 μl0.1% blue dextran (detected by color) in 100 mM ammonium formate buffer,pH 5, plus 200 mM NaCl (detected with AgNO₃). Samples were eluted with100 nM ammonium formate buffer, pH 5, into 500 μl fractions containingthe Vo(³H) cpm which were pooled, lyophilized, dissolved in H₂O, andstored at −20° C. SEPHADEX G-15 chromatography was used to removeadditional contaminants.

Selectin affinity columns having a ratio of 1.7 mg of selectin-Rg to 1ml PAS were prepared and used for the PIM-heparin studies. Aliquots of³H-labeled PIM-Heparin were applied to L-selectin, P-selectin orE-selectin affinity columns equilibrated in a buffer consisting of 20 mMHepes, 125 mM NaCl, 2 mM CaCl₂, 2 mM MgCl₂, pH 7.45 (Koenig et al.,supra, 1997). The column was washed and the bound PIM-Heparin elutedwith the same buffer containing 2 mM EDTA (divalent cation-dependentcomponent) in place of 2 mM CaCl₂ and 2 mM MgCl₂. The second elution waswith 20 mM Hepes (pH 7.45), 110 mM NaCl, 20 mM EDTA (divalentcation-independent component, EDTA acting as a polycarboxylate) and thefractions were collected as described above.

Using the L-selectin affinity column, 10% of the added PIM-heparin ranthrough, 58% of the heparin that bound to the column was eluted with 2mM EDTA and the remaining 32% was eluted with 20 mM EDTA (FIG. 4B). Incontrast, for the P-selectin column, 21% of the material ran through,very little was eluted with 2 mM EDTA, and the 79% that remained boundto the column required repeated washing with 20 mM EDTA for elution(FIG. 4C). For the E-selectin column, 100% of the PIM-heparin ranthrough (FIG. 4A).

These results demonstrate that the binding of PIM-heparin to theselectins is similar to that of the HS chains obtained from endothelialcells, except that a fraction of PIM-heparin remained bound toL-selectin after elution with 2 mM EDTA.

B. Calcium Dependence of Heparin Binding to L-selectin and to P-selectin

The calcium dependence of PIM-heparin binding to the L-selectin andP-selectin columns was determined as described above for endothelial HSchains. Aliquots of ³H-labeled PIM-Heparin were applied to an L-selectinand P-selectin affinity column in a magnesium/EGTA buffer having aresidual free calcium concentration of 774 μM. The columns were washed,eluted and the fractions were collected as described above.

L-selectin and P-selectin binding by PIM-heparin was similar to theendothelial HS chains (FIG. 5). For the L-selectin column, 100% ranthrough; for the P-selectin column, 55% ran through, 3% eluted with 2 mMEDTA, and 42% eluted with 20 mM EDTA. Thus, binding to L-selectin wasabolished in Mg++/EGTA buffers, in which calcium was present innegligible amounts. The fraction bound to P-selectin was lower (45% vs.79%) than that detected under calcium-replete conditions indicating apartial calcium dependency of the initial binding (compare FIG. 5 withFIG. 4). The fraction that did bind in the absence of calcium required20 mM EDTA for elution (FIG. 5B).

These results demonstrate that calcium assists the initial binding ofsome PIM-heparin fragments to P-selectin. However, once bound, calciumis not required to maintain the interaction of the heparin chains withP-selectin.

EXAMPLE III Binding and Characterization of Size-Defined HeparinFragments

This example shows that L-selectin and P-selectin exhibit differentialpreferences in their binding towards heparin fragments.

A. Binding of Size-fractionated Heparin Fragments to the SelectinColumns

The binding of sized fragments of heparin to L-selectin and P-selectinwas analyzed. Size-fragmented mixtures of PIM-heparin were prepared aspreviously described (Pervin et al., supra, 1995), labeled by reductionwith NaB(³H)₄ and purified as described above. Aliquots of the labeledPIM-heparin fragments were loaded onto the L-selectin and P-selectinaffinity columns, washed, and the bound ligand eluted and fractionscollected as described above. Again, L-selectin binding was eluted by 2mM EDTA, whereas most of the binding to P-selectin required 20 mM EDTAfor elution, although a small portion was eluted with 2 mM EDTA (seeTable 4).

The results shown in FIG. 6 and Table 2 indicate that the binding ofthese (³H)-heparin fragments to L-selectin and P-selectin is sizedependent; substantial binding of tetradecasaccharides was observed(FIG. 6D). Under the conditions of affinity column chromatography usedhere, binding constants in the low μM range are expected to be requiredfor detection of binding. Consistent with this prediction, thetetradecasaccharides have IC₅₀ values in the low uM range for inhibitionof L-selectin and P-selectin binding to SLe^(x) (Table 3). Thus, thetetradecasaccharide mixture has IC₅₀ values that are about 6-fold to10-fold greater than those reported for SLe^(x) itself(tetradecasaccharides: 82 μM and 54 μM for P-selectin and L-selectin,respectively; and SLe^(x): 520 μM and 600 μM for P-selection andL-selectin, respectively)o. Studies with Mg⁺⁺/EGTA buffers confirmed thecalcium requirement for L-selectin binding, indicating that the divalentcation dependence of this interaction was similar to that of bulkheparin and of the endothelial HS chains.

The fractionated (³H)-tetradecasaccharide heparin fragments werereapplied t o the L-select in and P-selectin columns to determine thereproducibility of the above results. In addition, cross-binding studieswere performed to identify differences in the tetradecasaccharides thatbound to L-selectin and P-selectin. (³H)-tetradecasaccharides(approximately 500,000 cpm) were loaded onto L-selectin and P-selectincolumns and the fractions collected as described above (only 0.5% ofeach fraction was monitored). Run-through and eluted fractions weredesalted using a CENTRICON unit (Amicon, Beverly Mass.) and the isolatedpools were stored in 250 μl H₂O at 4° C. Recoveries ranged from 50-99%.

For L-selectin, the pools were unbound (Pool A), slightly retarded (PoolB), retarded (Pool C) or eluted with EDTA (Pool D). For rebindingexperiments, the pools were reapplied (2500 cpm) to the same column. Forcross-binding experiments, (³H)-tetradecasaccharides (2500 cpm) thatbound to L-selectin were loaded onto the P-selectin column and the(³H)-tetradecasaccharides (2500 cpm) that bound to P-selectin wereloaded onto the L-selectin column. The columns were washed, eluted andthe fractions were collected as previously described.

Rebinding of the pooled tetradecasaccharide fractions to L-selectin andP-selectin indicated that the binding profiles were reproducible (FIG. 7and Table 4). In addition, 100% of the tetradecasaccharides that boundto L-selectin bound to P-selectin; these tetradecasaccharides wereeluted with 20 mM EDTA (Table 4). Only a subfraction of thetetradecasaccharides that bound P-selectin also bound to L-selectin: 22%of the 2 mM EDTA-eluted and 51% of the 20 mM EDTA elutedmaterial. Of thefraction that did not bind to L-selectin, 12% rebound to P-selectin andeluted from the column only with 20 mM EDTA.

These findings indicate that, while L-selectin and P-selectin bothexhibit some selectivity in recognizing heparin tetradecasaccharides,P-selectin has a more permissive range of recognition.

B. Analysis of the Structural Features of the Tetradecasaccharides thatDetermine Binding Behavior to P-selectin and L-selectin

The structural differences that account for differential binding wasinvestigated by determining the type of tetradecasaccharides that boundto L-selectin and P-selectin. Aliquots (2000 cpm) of the pooledfractions isolated from the L-selectin and P-selectin columns weresubjected to cleavage either with specific heparin lyases (I, II andIII) as previously described (Lohse and Linhardt, supra, 1992; Desai etal., supra, 1993; Jandik et al., supra, 1994; Linhardt, supra, 1996) orby nitrous acid treatment at pH 1.5 or pH 4.0 (Conrad, supra, 1996).These treatments result in the cleavage of heparin chains, the type ofwhich depends upon specific structural features or modifications of theheparin molecule.

Heparin lyase I (0.1-0.05 units) was used to digest(H)-tetradecasaccharide, and 0.05 units were used to digest each of thepools at 30° C. for 8.5 hours in sodium phosphate/NaCl buffer, pH 7.1.Heparin lyase II (0.1 units) was used to digest all samples for 8 hoursat 35° C. in sodium phosphate, buffer, pH 7.1. Heparin lyase III (0.2units) was used to digest all samples for 11 hours at 35° C. in sodiumphosphate buffer, pH 7.6. All digestions went to completion and werestopped by boiling for 5 minutes; samples were stored at −20° C.

Nitrous acid (HONO) degradation was performed by adding 100 u L ofeither the pH 1.5 or pH 4.0 HONO reagent to tubes containing the(³H)-tetradecasaccharide or each pooled fraction. The reaction proceededfor 10 minutes at room temperature, then was quenched with either 15 μlof 1 M Na₂CO₃ for the pH 4.0 reaction or 35 μl of 1 M Na₂CO₃ for the pH1.5 reaction to obtain a final pH of 7.5. pH controls for each reactionwere used for every experiment and consisted of replacing the sodiumnitrite with sodium acetate for the pH 4.0 reaction, and barium nitritewith barium acetate for the pH 1.5 reaction. Samples were stored at −20°C. until FPLC (forward phase liquid chromatography) analysis.

The size of the treated fractions were determined by FPLC using aSUPERDEX 75 HR 10/30 column (Pharmacia, Alameda Calif.). The(³H)-tetradecasaccharide starting mixture, and the L-selectin andP-selectin binding and nonbinding (³H)-tetradecasaccharide-pools wereanalyzed in parallel. The column was run isocratically in the samebuffer used for the selectin-Rg-PAS columns (20 mM Hepes (pH 7.45), 125mM NaCl, 2 mM MgCl₂, 2 mm CaCl₂). An FPLC system (Pharmacia, P-LKB-PumpP-500; P-LKB-Controller LCC-500 Plus; LKB-Frac 100) was used to elutethe column at 1.0 ml/minute flow rate and collect 0.25 minute fractionsafter 6 min from the beginning of the run to 21 min resulting in 60fractions per run being collected. The fractions were collected inscintillation vials and the radioactivity determined. The elutionprofile of this column was highly reproducible from run to run, asdetermined by addition of blue dextran (void column) and PIM-heparinsized mixtures ranging from disaccharides to tetradecasaccharides, whichwere radiolabeled with NaB(³H)₄ as described above. 50 μl of 1% bluedextran was added to every sample prior to loading onto the SUPERDEX 75FPLC column to confirm run to run reproducibility.

L-selectin binding fragments were sensitive to Heparin Lyase I and HONOpH 1.5 and, therefore, the fragments included the more heavily sulfatedand epimerized regions. The sensitivity to HONO, pH 4.0, treatmentindicates that the L-selectin binding heparin tetradecasaccharide chainsare enriched in the small amounts of free amino groups present in themixture. The P-selectin binding component includes this fraction, aswell as a fraction sensitive to Heparin Lyase III digestion whichindicates the presence of less heavily modified tetradecasaccharidechains.

EXAMPLE IV Heparin Inhibits L-Selectin and P-Selectin Binding

This example shows that pharmaceutical formulations of heparin caninhibit L-selectin and P-selectin binding.

A. Heparin Inhibits L-selectin and P-selectin Binding to SLe^(x)

The inhibitory properties of clinically formulated pharmaceuticalheparin preparations were studied using ELISA inhibition assays.

ELISA inhibition assays were performed as previously reported (O'Connellet al., Proc. Natl. Acad. Sci. USA 93:5883-5887 (1996); Koenig et al.,supra, 1997). Sterile 96 well ELISA plates were coated with 200 ng ofpolyacrylamide-SLe^(x) (Glycotech; Rockville Md.) by overnightincubation at 40° C. in 100 μl of 50 mM sodium carbonate/bicarbonatebuffer, pH 9.5. Plates were blocked for at least 2 hours at 4° C. with200 μl of 20 mM Hepes, 125 mM NaCl, 2 mM CaCl₂, 2 mM MgCl₂, 1%protease-free BSA, pH 7.45 (osmolarity 290 milliosmoles) per well.During the blocking step, the selectin-Rg chimeras were separatelypreincubated at 4° C. with a peroxidase-conjugated goat anti-human IgGsecondary antibody (Jackson Immunoresearch Laboratories, Inc.; WestGrove Pa.) in assay buffer for approximately 1 hour.

Final selectin-Rg concentration was 20 nM, and the secondary antibodydilution was 1:1000. Potential binding inhibitors were serially dilutedin assay buffer at twice the final required concentration. Theselectin-Rg/secondary antibody stock was aliquoted into tubes containingan equivalent volume of inhibitor solution; buffer, alone, for thepositive control; or buffer with 10 mM Na₂EDTA, pH 7.5, for the negativecontrol (final concentration 5 mM EDTA).

These tubes were preincubated at 4° C. for 30 minutes, then added toELISA plates, in duplicates, to a well volume of 100 μl. After 4 hoursat 4° C., the plates were washed three times with 200 μl per well ofassay buffer at 4° C., followed by development with 150 μl per well ofOLD solution (0.002 mg o-phenylenediamine dyhydrochloride (OPD)/ml in 50mM sodium citrate, 50 mM disodium phosphate buffer, 1 μl/ml 30% H₂O₂, pH5.2) at room temperature. Each well was sequentially quenched with 40 μlof 4 M H₂SO₄ after a fixed amount of time. The absorbance at 492 nM wasdetermined using SOFTMAX software and a microplate reader (MolecularDevices Inc., Menlo Park Calif.). Prior to curve fitting, the absorbancevalues were converted into percentages for comparative purposes usingthe formula: ({(average of duplicates)−(negative control)}/{(positivecontrol)−(negative control)})×100) using the SOFTMAX software.

Two separate lots of pharmaceutical unfractionated heparin significantlyinhibited L-selectin and P-selectin, but not E-selectin binding toimmobilized SLe^(x) (FIG. 8 and Table 5). The concentrations requiredwere less than the recommended therapeutic range for anticoagulation;IC₅₀ values of 0.07-0.08 units/ml and 0.01-0.02 units/ml towardsSLe^(x)-binding of L-selectin and P-selectin, respectively (Table 5). Inaddition, two different types of clinical grade LMW heparins, FRAGMINand LOVENOX, gave IC₅₀ values of 0.7-2.0 units/ml and 0.8-1.5 units/mlfor inhibition of L-selectin and 1.5-2.0 units/ml and 0.8-1.0 units/mlfor inhibition of P-selectin binding, respectively, which were at orhigher than the recommended therapeutic levels (see FIG. 8 and Table 5).

B. Heparin Inhibits L-selectin and P-selectin Binding to HL-60 Cells

The inhibitory properties of pharmaceutical heparin samples also werestudied using HL-60 cell attachment assays. HL-60, a human promyelocyticleukemia cell line (ATCC; CCL 240)), was grown in RPMI 1640 mediasupplemented with 20% FBS (1% Pen-Strep/0.2% gentamycin) and used at orbefore 8 passages. The binding inhibition studies were performed bylabeling HL-60 cells with (³H)-thymidine (1 μCi/ml media) for two days.After labeling, the cells, at a density of 2×10⁶ cells/ml or less, werecollected by centrifugation, washed three times with 20 ml of media,resuspended and the number of cells (1×10⁴ to 1×10⁵ cells/well) andradioactivity were determined (0.3-1.7 cpm/cell). 1 pmole per well ofL-selectin-Rg and P-selectin-Rg immobilized on 24 well tissue cultureplates in 250 μl of 50 mM sodium carbonate/bicarbonate buffer, pH 9.5,for 8 to 12 hours at 4° C. were blocked for 2 hours at 4° C. with 500 μlper well of 20 mM Hepes, 125 mM NaCl, 2 mM CaCl₂, 2 mM MgCl₂, and 1%protease-free BSA, pH 7.45.

Serial dilutions of unfractionated and LMW heparins were prepared induplicate as described above and aliquots of (³H)-HL-60 cells added fora final volume of 250 μl per well. Positive control wells with(³H)-HL-60 cells alone and negative controls including 20 mM EDTA wereanalyzed in triplicate. After incubation for 3-4 hours at 4° C. withgentle rotation, the wells were washed 3 times with 500 μl cold assaybuffer lacking BSA. Bound cells were solubilized at room temperature for10 minutes in 1% Triton X-100 and the lysate monitored forradioactivity. IC₅₀ values are expressed as a percentage of the positivecontrol and calculated as described above for the ELISA inhibitionexperiments (Table 5).

Unfractionated heparin inhibits the interaction of HL-60 with L-selectinand P-selectin (FIG. 9). The IC₅₀ values calculated are 0.02-0.03units/ml for L-selectin and 0.003-0.01 units/ml for P-selectin (Table5). These values are approximately 12-fold and 50-fold lower than therecommended therapeutic range for heparin anticoagulant therapy. Theseresults indicate that levels of heparin less than those used inanticoagulation therapy are effective at inhibiting L-selectin andP-selectin binding. The results also indicate that the two low molecularweight heparins (FRAGMIN and LOVENOX) are much poorer inhibitors ofL-selectin and P-selectin binding to sialylated ligands, including theHl-60 ligand PSGL-1 (FIG. 9 and Table 5).

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the spirit of the invention.

18. Albeida, S. M., C. W. Smith, and P. A. Ward. 1994. Adhesionmolecules and inflammatory injury. FASEB J. 8:504-512.

25. Tedder, T. F., D. A. Steeber, A. Chen, and P. Engel. 1995. Theselectins: Vascular adhesion molecules. FASEB J. 9:866-873.

57. Ridings, P. C., S. Holloway, G. L. Bloomfield, M. L. Phillips, B. J.Fisher, C. R. Blocher, H. J. Sugerman, and A. A. Fowler,III. 1997.Protective role of synthetic sialylated oligosaccharide insepsis-induced acute lung injury. J. Appl. Physiol. 82:644-651.

58. Mengelers, H. J. J., T. Maikoe, B. Hooibrink, T. W. Kuypers. J.Kreukniet, J.-W. J. Lammers. and L. Koenderman. 1993. Down modulation ofL-Selectin expression on cosinophils recovered from bronchoalveolarlavage fluid after allergen provocation. Clin. Exp. Allergy 23:196-204.

59. Georas, S. N., M. C. Liu, W. Newman, L. D. Beall, B. A. Stealey, andB, S. Bochner. 1992. Altered adhesion molecule expression andendothelial cell activation accompany the recruitment of humangranulocytes to the lung after segmental antigen challenge. Am. J.Respir. Cell Mol. Biol. 7:261-269.

60. Mulligan, M. S., S. R. Watson, C. Fennie, and P. A. Ward. 1993.Protective effects of selectin chimeras in neutrophil-mediated lunginjury. J. Immunol. 151:6410-6417.

61. Mulligan, M. S., M. Miyasaka, T. Tamatani, M. L. Jones, and P. A.Ward, 1994. Requirements for L-selectin in neutrophil-mediated lunginjury in rats. J. Immunol. 152:832-840.

62. Doyle, N. A., S. D. Bhagwan, B. B. Meek, G. J. Kutkoski, D. A.Steeber, T. F. Tedder, and C. M. Doerschuk. 1997. Neutrophilmargination, sequestration, and emigration in the lungs ofL-selectin-deficient mice. J. Clin. Invest. 99:526-533.

63. Mulligan, M. S., J. C. Paulson, S. De Frees, Z.-L. Zheng, J. B.Lowe, and P. A. Ward. 1993. Protective effects of oligosaccharides inP-selectin-dependent lung injury. Nature 364:149-151.

64. Mulligan, M. S., M. Miyasaka, Y. Suzuki, H. Kawashima, M. Iizuka. A.Hasegawa, M. Kiso, R. L. Warner, P. A. Ward, and T. Suzuki. 1995.Anti-inflammatory effects of sulfatides in selectin-dependent acute lunginjury [published erratum appears in Int Immunol 1995October;7(10):1699]. Int. Immunol. 7:1107-1113.

65. Burns, A. R. and C. M. Doerschuk. 1994. Quantitation of L-selectinand CD 18 expression on rabbit neutrophils during CD 18-independent andCD 18dependent emigration in the lung. J. Immunol. 153:3177-3188.

66. Mizgerd, J. P., B. B. Meek, G. J. Kutkoski, D. C. Bullard, A. L.Beaudet, and C. M. Doerschuk. 1996. Selectins and neutrophil traffic:Margination and Streptococcus pneumoniae-induced emigration in murinelungs. J. Exp. Med. 184:639645.

67. Seekamp, A., G. O. Till, M. S. Mulligan, S. C. Paulson, D. C.Anderson, M. Miyasaka, and P. A. Ward. 1994. Role of selectins in localand remote tissue injury following ischemia and reperfusion. Am. J.Pathol. 144:592-598.

68. Moore, T. M., P. Khimenko, W. K Adkins, M. Miyasaka, and A. E.Taylor. 1995. Adhesion molecules contrbute to ischemia andreperfusion-induced injury in the isolated rat lung. J. Appl. Physiol.78:2245-2252.

69. Ridings, P. C., A. C. J. Windsor, M. A. Jutila, C. R. Blocher, B. J.Fisher, M. M. Sholley, H. J. Sugerman, and A. A. Fowler,III. 1995. Adual-binding antibody to E- and L-selectin attenuates sepsis-inducedlung injury. Am. J. Respir. Crit. Care Med. 152:247-253.

70 Tedder. T. F., D. A. Steeber, and P. Pizcueta. 1995.L-selectin-deficient mice have impaired leukocyte recruitment intoinflammatory sites. J. Exp. Med. 181:2259-2264.

71. Catalina, M. D., M. C. Carroll, H. Arizpe, A. Takashima, P. Estess,and M. H. Siegelman. 1996. The route of antigen entry determines therequirement for L-selectin during immune responses. J. Exp. Med.184:2341-2351.

72. Narasinga Rao, B. N., M. B. Anderson J. H. Musser, J. H. Gilbert, M.E. Schaefer, C. Foxall, and B K Brandley 1994 Sralyl Lewis X mimicsderived from a pharmacophore search are selectin inhibitors withanti-inflammatory activity. J. Biol. Chem. 269:19663-19666.

73. Elbim, C., M. H. Prevor, F. Bouscarat, E. Franzini, S.Chollet-Martin, J: Hakim, and M. A. Gougerot-Pocidalo. 1994.Polymorphonuclear neutrophils from human immnunodeficiencyvirus-infected patients show enhanced activation, diminishedfMLP-induced L-selectin shedding, and an impaired oxidative burst aftercytokine priming. Blood 84:2759-2766.

74. Xu, J. C., I. S. Grewal, G. P. Geba, and R. A. Flavell. 1996.Impaired primary T cell responses in L-selectin-deficient mice. J. Exp.Med. 183:589-598.

75. Zhang, J. G., L. Morgan, and G. P. Spickett. 1996. L-selectin inpatients with common variable immunodeficiency (CVID): A comparativestudy with normal individuals. Clin. Exp. Immunol. 104:275-279.

76. Kawaishi, K., A. Kimura, O. Katoh, A. Sasaki, N. Oguma, A. Ihara,and Y. Satow. 1996. Decreased L-selectin expression in CD34-positivecells from patients with chronic myelocytic leukaemia. Br. J. Haematol.93:367-374.

77. Aziz, K. E., P. J. McCluskey, and D. Wakefield. 1996. Expression ofselectins (CD62 E,L,P) and cellular adhesion molecules in primarySjogren's syndrome: Questions to immunoregulation. Clin. Immunol.Immunopathol. 80:55-66.

78. Munro, J. M., D. M. Briscoe, and T. F. Tedder. 1996. Differentialregulation of leucocyte L-selectin (CD62L) expression in normal lymphoidand inflamed extralymphoid tissues. J. Clin. Pathol. 49:721-727.

79. Steeber, D. A., N. E. Green, S. Sato, and T. F. Tedder. 1996.Humoral immune responses in L-selectin-deficient mice. J. immunol.157:4899-4907.

80. Sweki, A., A.-S. Cordey, N. Monai, J.-C. De Flaugerues, M. Schapira,and O. Spertini. 1995. Cleaved L-selectin concentrations in meningealleukaemia. Lancet 345:286-289.

81. Hänninen, A., C. Taylor, P. R. Streeter, L. S. Stark, J. M. Sarte,J. A. Shizuru, O. Simell, and S. A. Michie. 1993. Vascular addressinsare induced on islet vessels during insulitis in nonobese diabetic miceand are involved in lymphoid cell binding to islet endothelium. J. Clin.Invest. 92:2509-2515.

82. Yang, X.-D., N. Karin, R. Tisch, L. Steinman, and H. O. McDevitt.1993. Inhibition of insulitis and prevention of diabetes in nonobesediabetic mice by blocking L-selectin and very late antigen 4 adhesionreceptors. Proc. Natl. Acad. Sci. USA 90:10494-10498.

83. Lepault, F., M.-C. Gagnerault, C. Faveeuw, H. Bazin, and C. Boitard.1995. Lack of L-selectin expression by cells transferring diabetes inNOD mice: Insights into the mechanisms involved in diabetes preventionby Mel-14 antibody treatment, Eur. J. Immunol, 25:1502-1507.

84. Hosaka, S., M. R. Shah, R. M. Pope, and A. E. Koch. 1996. Solubleforms of P-selectin and intercellular adhesion molecule-3 in synovialfluids. Clin. Immunol. Immunopathol. 78:276-282.

85. Blann, A. D., P. A. Sanders, A. Herrick, and M. I. V. Jayson. 1996.Soluble L-selectin in the connective tissue diseases. Br. J. Haematol.95:192-194.

86. Inagaki, H., K. Suzuki, K. Nomoto, and Y. Yoshikai. 1996. Increasedsusceptibility to primary infection with Listeria monocytogenes ingermfree mice may be due to lack of accumulation of L-selectin⁺ CD44⁺ Tcells in sites of inflammation. Infect. Immun. 64:3280-3287.

87. Kajihara, J., Y. Guoji, K. Kato, and Y. Suzuki. 1995. Sulfatide, aspecific sugar ligand for L-selectin, blocks CCl₄-induced liverinflammation in rats. Biosci. Biotechnol. Biochem. 59:155-157.

88. Wada, Y., T. Saito, N. Matsuda, H. Ohmoto, K. Yoshino, M. Ohashi, H.Kondo, H. Ishida, M. Kiso, and A. Hasegawa. 1996. Studies on selectinblockers .2. Novel selectin blocker as potential therapeutics forinflammatory disorders. J. Med. Chem. 39:2055-2059.

89. Mihelcic, D., B. Schleiffenbaum, T. F. Tedder, S. R. Sharar, J. M.Harlan, and R. K Winn. 1994. Inhibition of leukocyte L-selectin functionwith a monoclonal antibody attenuates reperfusion injury to the rabbitear. Blood 84:2322-2328.

90. Han, K. T., S. R. Sharar, M. L. Phillips, J. M. Harlan, and R. K.Winn. 1995. Sialyl Lewis^(x) oligosaccharide reducesischemia-reperfusion injury in the rabbit ear. J. Immunol. 155:4011-4015.

91. McGill, S. N., N. A. Ahmed, F. Hu, R. P. Michel, and N. V. Christou.1996. Shedding of L-selectin as a mechanism for reducedpolymorphonuclear neutrophil exudation in patients with the systemicinflammatory response syndrome. Arch. Surg. 131:1141-1146.

92. Ahmed, N. A. and N. V. Christou. 1996. Decreased neutrophilL-selectin expression in patients with systemic inflammatory responsesyndrome. Clin Invest. Med. 19:427-434.

93. Cecconi, O., R. M. Nelson, W. G. Roberts, K. Hanasaki, G. Mannori,C. Schulz, T. R. Ulich, A. Aruffo, and M. P Bevilacqua. 1994. Inositolpolyanions. Noncarbohydrate inhibitors of L- and P-selectin that blockinflammation. J. Biol. Chem. 269:15060-15066.

94. Briggs. J. B., Y. Oda, J. H. Gilbert, M. E. Schaefer, and B. A.Macher. 1995. Peptides inhibit selectin-mediated cell adhesion in vitro,and neutrophil influx into inflammatory sites in vivo. Glycobiology5:583-588.

95. Mannori, G., P. Crottet. O. Cecconi, K. Hanasaki, A. Aruffo, R. M.Nelson, A. Varki, and M. P. Bevilacqua. 1995. Differential colon cancercell adhesion to E-, P-, and L-selectin: Role of mucin typeglycoproteins. Cancer Res. 55:4425-4431.

96. Kawabata, K., Y. Nagake, K. Shikata, H. Makino, and Z. Ota. 1996.The changes of Mac-1 and L-selectin expression on granulocytes andsoluble L-selectin level during hemodialysis. Nephron 73:573-579.

97. Rabb, H., G. Ramirez, S. R. Saba, D. Reynolds, J. C. Xu, R. Flavell,and S. Antonia. 1996. Renal ischemic-reperfusion injury inL-selectin-deficient mice. Am. J. Physiol. Renal,Fluid ElectrolyrePhysiol. 271:F408-F413.

98. Ley, K., G. Linnemann, M. Meinen, L. M. Stoolman, and P. Gaehtgens.1993. Fucoidin, but not yeast polyphosphomannan PPME, inhibits leukocyterolling in venules of the rat mesentery. Blood 81:177-185.

99. Kuijper, P. H. M., H. I. G. Torres, J. A. M. Van der Linden, J. W.I. Lammers, J. J. Sixma, L. Koenderman, and J. J. Zwaginga. 1996.Platelet-dependent primary hemostasis promotes selectin- andintegrin-mediated neutrophil adhesion to damaged endothelium under flowconditions. Blood 87:3271-3281.

100. Buerke, M., A. S. Weyrich, Z. Zheng, F. C. A. Gaeta, M. J. Forrest,and A. M. Lefer. 1994. Sialyl Lewis^(x)-containing oligosaccharideattenuates myocardial reperfusion injury in cats. J. Clin. Invest.93:1140-1148.

101. Miura, T., D. P. Nelson, M. L. Schermerhorn, T. Shin'oka, G. Zund,P. R. Hickey, E. J. Neufeld, and J. E. Mayer,Jr., 1996. Blockade ofselectin-mediated leukocyte adhesion improves postischemic function inlamb hearts. Ann. Thorac. Surg. 62:1295-1300.

102. Flynn, D. M., A. I. Buda, P. R. Jeffords, and D. J. Lefer. 1996.Sialyl Lewis^(x)-containing carbohydrate reduces infarct size: Role ofselectins in myocardial reperfusion injury. Am. J. Physiol. Heart Circ.Physiol. 271:H2086-H2096.

103. Ma, X., A. S. Weyrich, D. J. Lefer, M. Buerke, K. H. Albertine, T.K. Kishimoto, and A. M. Lefer. 1993. Monoclonal antibody to L-selectinattenuates neutrophil accumulation and protects ischemic reperfused catmyocardium. Circulation 88:649-658.

104. Buerke, M., A. S. Weyrich, T. Murohara, C. Queen, C. K. Klingbeil,M. S. Co, and A. M. Lefer. 1994. Humanized monoclonal antibody DREG-200directed against L-selectin protects in feline myocardial reperfusioninjury. J. Pharmacol. Exp. Ther. 271:134-142.

105. Lefer, A. M. 1995. Role of selectins in myocardial ischemiareperfusion injury. Ann. Thorac Surg. 60:773-777.

106. Haught, W. H., M. Mansour, R. Rothlein, T. K. Kishimoto, E. A.Mainolfi, J. B. Hendricks, C. Hendricks, and J. L. Mehta 1996.Alterations in circulating intercellular adhesion molecule-1 andL-selectin: Further evidence for chronic inflammation in ischernic heartdisease. Am. Heart J. 132:1-8.

107. Murohara, T., M. Buerke, and A. M. Lefer. 1994. Polymorphonuclearleukocyte-induced vasocontraction and endothelial dysfunction: Role ofselectins. Arterioscler. Thromb. 14:1509-1519.

108. Ramamoorthy, C., S. R. Sharar, J. M. Harlan, T. F. Tedder, and R.K. Winn. 1996. Blocking L-selectin function attenuates reperfisioninjury following hemorrhagic shock in rabbits. Am. J. Physiol. HeartCirc. Physiol. 271 :H1871-H1877.

109. Blann, A., J. Morris, and C. McCollum. 1996. Soluble L-selectin inperipheral arterial disease: Relationship with soluble E-selectin andsoluble P-selectin. Atherosclerosis 126:227-231.

110. Turunen, J. P., M. L. Majuri, A. Seppo, S. Tiisala, T. Paavonen, M.Miyasaka, K. Lemström, L. Pentril{haeck over (a)}, O. Renkonen. and R.Renkonen, 1995. De novo expression of endothelial sialyl Lewis^(a) andsialyl Lewis^(x) during cardiac transplant rejection: Superior capacityof a tetravalent sialyl Lewis^(x) oligosaccharide in inhibitingL-selectin-dependent lymphocyte adhesion. J. Exp. Med. 182:1133-1141.

111. Bargatze, R. F., S. Kurk, G. Watts, T. K. Kishimoto, C. A Speer,and M. A. Jutila, 1994. In vivo and in vitro functional examination of aconserved epitope of L- and E-selectin crucial for leukocyte-endothelialcell interactions. J. Immunol. 152:5814-5825.

112. Granert, C. J. Raud, X. Xie, L. Lindquist, and L. Lindbom. 1994.Inhibition of leukocyte rolling with polysaccharide fucoidin preventspleocytosis in experimental meningitis in the rabbit. J. Clin. Invest.93:929-936.

113. Dopp, J. M., S. M. Breneman, and J. A. Olschowka. 1994. Expressionof ICAM-1, VCAM-1, L-selectin, and leukosialin in the mouse centralnervous system during the induction and remission stages of experimentalallergic encephalomyclitis. J. Neuroimmunol. 5:129-144

114. Bührer, C., R. Harold, D. Stibenz, G. Henze, and M. Obladen. 1996.Cerebrospinal fluid soluble L-selectin (sCD62L) in meningoencephalitis.Arch. Dis. Child. 74:288-292.

115. Morikawa, E., S. M. Zhang, Y. Seko, T. Toyoda, and T. Kirino. 1996.Treatment of focal cerebral ischemia with synthetic oligopeptidecorresponding to lectin domain of selectin. Stroke 27:951-955.

116. Mössner, R., K. Fassbender. J. Kühnen, A. Schwartz, and M.Hennerici. 1996. Circulating L-selectin in multiple sclerosis patientswith active, gadolinium-enhancing brain plaques. J. Neuroimmnunol.65:61-65.

117. Onrust, S. V., P. M. Hartl, S. D. Rosen, and D. Hanahan. 1996.Modulation of L-selectin ligand expression during an immune responseaccompanying tumorigenesis in transgenic mice. J. Clin. Invest.97:54-64.

118. Wenisch, C., E. Presterl, W. Graninger, and S. Looarcesuwan. 1995.Circulating L-seletin is elevated in patients with Plasmodium falciparummalaria. J. Infect. Dis. 171:1078.

119. Rebuck, N., A. Gibson, and A. Finn. 1995. Neutrophil adhesionmolecules in term and premature infants: Normal or enhanced leucocytcintegrins but defective L-selectin expression and shedding. Clin. Exp.Immunol. 101:183-189.

120. Bührer, C., D. Stibenz, J. Graulich, U. Gernhold, E. C. Butcher, J.W. Dudenhausen, and M. Obladen. 1995. Soluble L-selectin (sCD62L)umbilical cord plasma levels increase with gestational age. Pediatr.Res. 38:336-341.

121. Wenisch, C., D. Myskiw, A. Gessi, and W. Graninger. 1995.Circulating selectins, intercellular adhesion molecule-1, and vascularcell adhesion molecule-1 in hyperthyroidism. J. Clin. Endocrinol. Metab.80:2122-2126.

122. Mulligan, M. S., M. J. Polley, R. J. Bayer, M. F. Nunn, J. C.Paulson, and P. A. Ward. 1992. Neutrophil-dependent acute lung injury.Requirement for P-selectin (GMP-140). J. Clin. Invest. 90:1600-1607.

123. Carden, D. L., J. A. Young, and D. N. Granger. 1993. Pulmonarymicrovascular injury after intestinal ischemia-reperfusion: Role ofP-selectin. J. Appl. Physiol. 75:2529-2534.

124. Shenkar, R., A. J. Cohen, D. Vestweber, Y. E. Miller, R. Tuder, andE. Abraham. 1995. Hemorrhage and resuscitation alter the expression ofICAM-1 and P-selectin in mice. Circ. Shock 45:248-259.

125. Kushimoto, S., K. Okajima, M. Uchiba, K. Murakami, H. Okabe, andK., Takatsuki. 1996. Pulmonary vascular injury induced by hemorrhagicshock is mediated by P-selectin in rats. Thromb. Res. 82:97-106.

126. Doerschuk, C. M., W. M. Quinlan. N. A. Doyle, D. C. Bullard, D.Vestweber, M. L. Jones, F. Takei, P. A. Ward, and A. L. Beaudet. 1996.The role of P-selectin and ICAM-1 in acute lung injury as determinedusing blocking antibodies and mutant mice. J. Immunol. 157:4609-4614.

127. Sakamaki, F., A. Ishizaka, M. Handa, S. Fujishima, T. Urano, K.Sayarna, H. Nakamura, M. Kanazawa, T. Kawashiro, M. Katayama, and Y.Ikeda. 1995. Soluble form of P-selectin in plasma is elevated in acutelung injury. Am. J. Respir. Crit. Care Med. 151:1821-1826.

128. De Sanctis, G. T., W. W. Wolyniec, F. H. Y. Green, S. X. Qin, A. P.Jiao, P. W. Finn, T. Noonan, A. A. Joetham, E. Gelfand, C. M. Doerschuk,and J. M. Drazen. 1997. Reduction of allergic airway responses inP-selectin-deficient mice. J. Appl. Physiol. 83:681-687.

129. Zeb, T., B. Piedboeuf, M. Gamache, C. Langston, and S. E. Welty.1996. P-Selectin is upregulated early in the course of hyperoxic lunginjury in mice. Free Radic. Biol. Med. 21:567-574.

130. Pottratz, S. T., T. D. Hall, W. M. Scribner, H. N. Jayaram, and V.Natarajan. 1996. P-selectin-mediated attachment of small cell lungcarcinoma to endothelial cells. Am. J. Physiol. Lung Cell. Mol. Physiol.271:L918-L923.

131. Ohkawara, Y., K. Yamauchi, N. Maruyama, H. Hoshi, I. Ohno, M.Honma, Y. Tanno, G. Tamara, K. Shirato, and H. Ohtani, 1995. In situexpression of the cell adhesion molecules in bronchial tissues fromasthmetics with air flow limitation: in vivo evidence of VCAM-1/VLA-4interaction in selective eosinophil infiltration. Am. J. Respir. CellMol. Biol. 12:4-12.

132. Henriques, G. M. O., J. M. Miotla, R. S. B. Cordeiro, B. A.Wolitzky, S. T. Woolley, and P. G. Hellewell. 1996. Selectins mediateeosinophil recruitment in vivo: A comparison with their role inneutrophil influx. Blood 87:5297-5304.

133. Mayadas, T. N. 1995. Gene knockout on P-selectin: Its biology andfunction. Trends Cardiovasc. Med. 5:149-157.

134. Simons, R. K., D. B. Hoyt, R. J. Winchell, R. M. Rose, and T.Holbrook. 1996. Elevated selectin levels after severe trauma: A markerfor sepsis and organ failure and a potential target for immunomodulatorytherapy. J. Trauma Injury Infect. Crit. Care 41:653-662.

135. Hansbrough, J. F., T. Wikström, M. Braide, M. Tenenhaus, O. H.Rennekampff, V. Kiessig, R. Zapata-Sirvent, and L. M. Bjursten. 1996.Effects of E-selectin and P-selectin blockade on neutrophilsequestration in tissues and neutrophil oxidative burst in burned rats.Crit. Care Med. 24:1366-1372.

136. Philips, M. L., B. R. Schwartz, A. Etzioni, R. Bayer, H. D. Ochs,J. C. Paulson, and J. M. Harlan. 1995. Neutrophil adhesion inleukocyteadhesion deficiency syndrome type 2. J. Clin. Invest. 96:2898-2906.

137. Bullard, D. C., E. J. Kunkel, H. Kubo, M. J. Hicks, I. Lorenzo, N.A. Doyle, C. M. Doerschuk, K. Ley, and A. L. Beaudet. 1996. Infectioussusceptibility and severe deficiency of leukocyte rolling andrecruitment in E-selectin and P-selectin double mutant mice. J. Exp.Med. 183:2329-2336.

138. Ohnishi, M., H. Koike, N. Kawamura, S. J. Tojo, M. Hayashi, and S.Morooka. 1996. Role of P-selectin in the early stage of the Anhusreaction. Immunopharmacology 34:161-170.

139. Austrup, F., D. Vestweber, E. Borges, M. Löhning, R. Bräuer, U.Hen, H. Renz, R. Hallmann, A. Scheffold, A. Radbruch, and A. Hamann.1997. P- and E-selectin mediate recruitment of T-helper-1 but notT-helper-2 cells into inflamed tissues. Nature 385:81-83.

140. Borges, E., W. Tietz, M. Steegmaier, T. Moll, R. Hallmann, A.Hamann, and D. Vestweber. 1997. P-selectin glycoprotein ligand-1(PSGL-1) on T helper 1 but not on T helper 2 cells binds to P-selectinand supports migration into inflamed skin. J. Exp. Med. 185:573-578.

141. Johnson, R. C., T. N. Mayadas, P. S. Frenette, R. E. Mebius, M.Subramaniam, A. Lacasce, R. O. Hynes, and D. D. Wagner. 1995. Blood celldynamics in P-selectin-deficient mice. Blood 86:1106-1114.

142. Bruserud, O., P. E. Akselen, J. Bergheim, and I. Nesthus. 1995.Serum concentrations of E-selectin, P-selectin, ICAM-1 and interleukin 6in acute leukaemia patients with chemotherapy-induced leucopenia andbacterial infections. Br. J. Haematol. 91:394-402.

143. Frenette, P. S., T. N. Mayadas, H. Rayburn, R. O. Hynes, and D. D.Wagner. 1996. Susceptibility to infection and altered hematopoiesis inmice deficient in both P- and E-selectins. Cell 84:563-574.

144. Jilma, B., P. Fasching, C. Ruthner, A. Rumplmayr, S. Ruzicka, S.Kapiotis, O. F. Wagner, and H. G. Eichler. 1996. Elevated circulatingP-selectin in insulin dependent diabetes mellitus. Thromb. Haemost.76:328-332.

145. Salmi, M., P. Rajala, and S. Jalkanen. 1997. Homing of mucosalleukocytes to joints-Distinct endothelial ligands in synovium mediateleukocyte-subtype specific adhesion. I. Clin. Invest. 99:2165-2172.

146. Walter, U. M. and A. C. Issekutz. 1997. The role of E- andP-selectin in neutrophil and monocyte migration in adjuvant-inducedarthritis in the rat. Eur. J. Immunol. 27:1498-1505.

147. Weiser, M. R., S. A. L. Gibbs, C. R. Valeri, D. Shepro, and H. B.Hechiman. 1996. Anti-selectin therapy modifies skeletal muscle ischemiaand reperfusion injury. Shock 5:402-407.

148. Nolte, D., P. Schmid, U. Jäger, A. Botzlar, F. Roesken, R. Hecht,E. Uhl, K. Messmer. and D. Vestweber. 1994. Leuckocyte rolling invenules of striated muscle and skin is mediated by P-selectin. not byL-selectin. Am. J. Physiol. Heart Circ. Physiol. 267:H1637-H1642.

149. Garcia-Criado, F. J., L. H. Toledo-Pereyra, F. Lopez-Neblina, M. L.Phillips, A. Paez-Rollys, and K. Misawa. 1995. Role of P-selectin intotal hepatic ischemia and reperfusion. J. Am. Coll. Surgeons181:327-334.

150. Rubio Avilla, J., J. M. Palma-Vargas, J. T. Collins, R. Smejkal, J.McLaren, L. M. Phillips, and L. H. Toledo-Pereyia. 1997. SialylLewis^(X) analog improves liver function by decreasing neutrophilmigration after hemorrhagic shock. J. Trauma Injury Infect. Crit. Care43:313-318.

151 Silber, A. W. Newman, K. A. Reimann, E. Hendricks, D. Walsh, and D.J. Ringler. 1994. Kinetic expression of endothelial adhesion moleculesand relationship to leukocyte recruitment in two cutaneous models ofinflammation. Lab. Invest. 70:163-175.

152. Winn, R. K., D. Liggitt, N. B. Vedder, J. C. Paulson, and J. M.Harian. 1993. Anti-P-selectin monoclonal antibody attenuates reperfusioninjury to the rabbit ear. J. Clin. Invest. 92:2042-2047.

153. Yamada, S., T. N. Mayadas, F. Yuan, D. D. Wagner, R. O. Hynes, R.J. Melder, and R. K. Jain. 1995. Rolling in P-selectin deficient mice isreduced but not eliminated in the dorsal skin. Blood 86:3487-3492.

154. Staite, N. D., J. M. Justen, L. M. Sly, A. L. Beaudet, and D. C.Bullard. 1996. Inhibition of delayed-type contact hypersensitivity inmice deficient in both E-selectin and P-selectin. Blood 88:2973-2979.

155. Subramaniam, M., S. Saffaripour, L. Van De Water, P. S. Frenette,T. N. Mayadas, R. O. Hynes, and D. D. Wagner. 1997. Role of endothelialselectins in wound repair. Am. J. Pathol. 150:1701-1709.

156. Kurose, I., T. Yamada, R. Wolf, and D. N. Granger. 1994.P-selectin-dependent leukocyte recruitment and intestinal mucosal injuryinduced by lactoferrin. J. Leukocyte Biol. 55:771-777.

157. Sun, X. M., R. A. Rozenfeld, X. W. Qu, W. Huang, F.Gonzalez-Crussi, and W. Hsueh. 1997. P-selectin-deficient mice areprotected from PAF-induced shock, intestinal injury, and lethality. Am.J. Physiol. Gastrointest. Liver Physiol. 273:G56-G61.

158. Mayadas, T. N., R. C. Johnson, H. Rayburn, R. O. Hynes, and D. D.Wagner. 1993. Leukocyte rolling and extravasation are severelycompromised in P selectin-deficient mice. Cell 74:541-554.

159. Kubes, P. and S. Kanwar. 1994. Histamine induces leukocyte rollingin post-capillary venules: A P-selectin-mediated event. J. Immunol.152:3570-3577.

160. Kubes, P., I. Kurose, and D. N. Granger. 1994. NO donors preventintegrin-induced leukocyte adhesion but not P-selectin-dependent rollingin postischemic venules. Am. J. Physiol. Heart Circ. Physiol.267:H931-H937.

161. Gibbs, S. A. L., M. R. Weiser, L. Kobzik, C. R. Valeri, D. Shepro,and H. B. Hechtman. 1996. P-selectin mediates intestinal ischemic injuryby enhancing complement deposition. Surgery 119:652-656.

162. Schürmann, G. M., A. E. Bishop, P. Facer, M. Vecchio, J. C. W. Lee,D. S. Rampton, and J. M. Polak. 1995. Increased expression of celladhesion molecule P-selectin in active inflammatory bowel disease. Gut36:411-418.

163. Bullard, D. C., L. Qin, I. Lorenzo, W. M. Quinlin, N. A. Doyle, R.Bosse, D. Vestweber, C. M. Doerschuk, and A. L. Beaudet. 1995.P-selectin/ICAM-1 double mutant mice: Acute emigration of neutrophilsinto the peritoneum is completely absent but is normal into pulmonaryalveoli. J. Clin. Invest. 95:1782-1788.

164. Panés, J., D. C Anderson, M. Miyasaka, and D. N. Granger. 1995.Role of leukocyce-endothelial cell adhesion in radiation-inducedmicrovascular dysfunction in rats. Gastroenterology 108:1761-1769.

165. Suzuki, Y., H. Ohtani, T. Mizoi, S. Takeha, K. Shiiba, S. Matsuno,and H. Nagura. 1995. Cell adhesion molecule expression by vascularendothelial cells as an immune/inflammatory reaction in human coloncarcinoma. Jpn. J. Cancer Res. 86:585-593.

166. Arndt, H., K. D. Palitzsch, D. C. Anderson, J. Rusche, M. B.Grisham, and D. N. Granger. 1995. Leucocyte-endothelial cell adhesion ina model of intestinal inflammation. Gut 37:374-379.

167. Tipping, P. G., X. R. Huang, M. C. Berndt, and S. R. Holdsworth.1994. A role for P selectin in complement-independentneutrophil-mediated glomerular injury. Kidney Int. 46:79-88.

168. Tipping, P. G., X. R. Huang, M. C. Berndt, and S. R. Holdsworth.1996. P-selectin directs T lymphocyte-mediated injury in delayed-typehypersensitivity responses: Studies in glomerulonephritis and cutaneousdelayed-type hypersensitivity. Eur. J. Immunol. 26:454-460.

169. Takada, M., K. C. Nadeau, G. D. Shaw, K. A. Marquette, and N. L.tilney. 1997. The cytokine-adhesion molecule cascade inischemia/reperfusion injury of the rat kidney—Inhibition by a solubleP-selectin ligand. J. Clin. Invest. 99:2682-2690.

170. Zizzi, H. C., G. B. Zibari, D. N. Granger, I. Singh, L. D. Cruz, F.Abreo, J. C. McDonald, and M. F. Brown. 1997 Quantification ofP-selectin expression after renal ishemia and reperfusion J. Pediatr.Surg. 32:1010-1013.

171 Suematsu, M., H. Suzuki, T. Tamatini, Y. Iigou, F. A. DeLano, M.Miyasaka, M. J. Forrest, R. Kannagi, B. W. Zweifach, Y. Ishimura, and G.W. Schmid-Schönbein. 1995. Impairment of selectin-mediated leukocytcadhesion to venular endothelium in spontaneously hypenensive rats. J.Clin. Invest. 96:2009-2016.

172. Davenpeck, K. L., T. W. Gauthier, K. H. Albertine, and A. M. Lefer.1994. Role of P-selectin in microvascular leukocyte-endothelialInteraction in splanchnic ischemia-reperfusion. Am. J. Physiol. HeartCirc. Physiol. 267:H622-H630.

173. Gauthier, T. W., K. L. Davenpeck, and A. M. Lefer. 1994. Nitricoxide attenuates leukocyte-endothelial interaction via P-selectin insplanchnic ischemia-reperfusion. Am. J. Physiol. Gastrointest. LiverPhysiol. 267:G562-G568.

174. Weyrich, A. S., X. Ma, D. J. Lefer, K. H. Albertine, and A. M.Lefer. 1993. In vivo neutralization of P-selectin protects feline heartand endothelium in myocardial ischemia and reperfusion injury. J. Clin.Invest. 91:2620-2629.

175. Lefer, D. J., D. M. Flynn, M. L. Phillips, M. Ratcliffe, and A. J.Buda. 1994. A novel sialyl Lewis^(x) analog attenuates neutrophilaccumulation and myocardial necrosis after ischeria and reperfsion.Circularion 90:2390-2401.

176. Lefer, D. J., D. M. Flynn, and A. I. Buda. 1996. Effects of amonoclonal antibody directed against P-selectin after myocardialischemia and reperfusion. Am. J. Physiol. Heart Circ. Physiol.270:H88-H98.

177. Scalia, R., T. Murohara, J. A. Delyani, T. O. Nossuli, and A. M.Lefer. 1996. Myocardial protection by N,N,N-trimethylsphingosine inischemia reperfusion injury is mediated by inhibition of P-selectin. J.Leukocyte Biol. 59:317-324.

178. Tojo, S. J., S. Yokota, H. Koike, J. Schultz, Y. Hamazume, E.Misugi, K. Yamada, M. Hayashi, J. C. Paulson, and S. Morooka. 1996.Reduction of rat myocardial ischemia and reperfusion injury by sialylLewis x oligosaccharide and anti-rat P-selectin antibodies. Glycobiology6:463-469.

179. Lefer, D. J., D. M. Flynn, D. C. Anderson, and A. J. Buda 1996.Combined inhibition of P-selectin and ICAM-1 reduces myocardial injuryfollowing ischemia and reperfusion. Am. J. Physiol. Heart Circ. Physiol.271:H2421-H2429.

180. Chignier, E., M. Parise, L. McGregor, C. Delabre, S. Faucompret,and J. McGregor. 1994. A P-selectin/CD62P monoclonal antibody (LYP-20),in tandem with flow cytometry, detects in vivo activated circulating ratplatelets in severe vascular trauma Thromb. Haemost. 72:745-749.

181. Lehr, H.-A., A. M. Olofsson, T. E. Carew, P. Vajkoczy, U. H. VonAndrian, C. Hübner, M. C. Berndt, D. Steinberg, K. Messmer, and K. E.Arfors. 1994. P-selectin mediates the interaction of circulatingleukocytes with platelets and microvascular endothelium in response tooxidized lipoprotein in vivo. Lab. Invest. 71:380-386.

182. Palabrica, T., R. Lobb, B. C. Furie, M. Aronovitz, C. Benjamin,Y.-M. Hsu. S. A. Sajer, and B. Furie. 1992. Leukocyte accumulationpromoting fibrin deposition is mediated in vivo by P-selectin onadherent platelets. Nature 359:848-851.

183. Winn, R. K., J. C. Paulson, and J. M. Harlan. 1994. A monoclonalantibody to P-selectin ameliorates injury associated with hemorrhagicshock in rabbits. Am. J. Physiol. Heart Circ. Physiol. 267:H2391-H2397.

184. Toombs, C. F., C. L. DeGraaf, J. P. Martin, J. G. Geng, D. C.Anderson, and R. J. Shebuski. 1995. Pretreatment with a blockingmonoclonal antibody to P-selectin accelerates pharmacologicalthrombolysis in a primate model of arterial thrombosis. J. Pharmacol.Exp. Ther. 275:941-949.

185. Fujise, K., B. M. Revelle, L. Stacy, E. L. Madison, E. T. H. Yeh,J. T. Willerson, and P. J. Beck. 1997. A tissue plasminogenactivator/P-selectin fusion protein is an effective thrombolytic agent.Circulation 95:715-722.

186. Lip, G. Y. H., A. D. Blann, J. Zarifis, M. Beevers, P. L. Lip, andD. G. Beevers. 1995. Soluble adhesion molecule P-selectin andendothelial dysfunction in essential hypertension: Implications foratherogenesis? A preliminary report. J. Hypertens. 13:1674-1678.

187. Subramaniatn, M., P. S. Frenette, S. Saffaripour, R. C. Johnson, R.O. Hynes, and D. D. Wagner, 1996. Defects in hemostasis inP-selectin-deficient mice. Blood 84:1238-1242.

188. Mazurov, A. V., D. V. Vinogradov, S. G. Khaspekova, A. V.Krushinsky, L. V. Gerdeva, and S. A. Vasthev. 1996. Deficiency ofP-selectin in a patient with grey platelet syndrome Eur. J. Heamatol57:38-41.

189. Close, C., M. Seigneur, M. Renard, and A. Pruvost. 1996. Influenceof hypoxia and hypoxiareoxygenation on endothelial P-selectinexpression. Haemostasis 26 Suppl. 4:177-181.

190. Closse, C., M. Seigneur, M. Renard, A. Pruvost, P. Dumain, F.Belloc, and M. R. Boisseau. 1997. Influence of hypoxia andhypoxia-reoxygenation on endothelial P-selectin expression. Thromb. Res.85:159-164.

191. Koskinen, P. K. and K. B. Lemström. 1997. Adhesion moleculeP-selectin and vascular cell adhesion molecule-1 in enhanced heartallograft arteriosclerosis in the rat. Circulation 95:191-196.

192. Sakai, A., N. Kume, E. Nishi, K. Tanoue, M. Miyasaka, and T. Kita.1997. P-selectin and vascular cell adhesion molecule-1 are focallyexpressed in aortas of hypercholesterolemic rabbits before intimalaccumulation of macrophages and T lymphocytes. Arterioscler. Thromb.Vasc. Biol. 17:310-316.

193. Tenaglia, A. N., A. J. Buda, R. G. Wilkins, M. K. Barron, P. R.Jeffords, K. Vo, M. O. Jordan, B. A. Kusnick, and D. J. Lefer. 1997.Levels of expression of P-selectin, E-selectin, and intercellularadhesion molecule-1 in coronary atherectomy specimens from patients withstable and unstable angina pectoris. Am. J. Cardiol. 79:742-747.

194. Ikeda, H., Y. Takajo, K. Ichiki, T. Ueno, S. Maki, T. Noda, K.Sugi. and T. Imaizuma. 1995. Increased soluble form of P-selectin inpatients with unstable angina. Circulation 92:1693-1696.

195. Kaikita, K., H. Ogawa, H. Yasue, T. Sakamoto, H. Suefuji, H.Sumida, and K. Okumura 1995. Soluble P-selectin is released into thecoronary circulation after coronary spasm. Circulation 92:1726-1730.

196. Tang, T., P. S. Frenette, R. O. Hynes, D. D. Wagner, and T. N.Mayadas. 1996. Cytokine-induced meningitis is dramatically attenuated inmice deficient in endothelial selectins. J. Clin. Invest. 97:2485-2490.

197. Whitcup, S. M., A. T. Kozhich, M. Lobanoff, B. A. Wolitzky, and C.C. Chan. 1997. Blocking both E-selectin and P-selectin inhibitsendotoxin-induced leukocyte infiltration into the eye. Clin. Immunnol.Immunopathol. 83:45-52.

198. Suzuki, H., B. W. Zweifach, M. J. Forrest, and G. W.Schmid-Schönbein. 1995. Modification of leukocyte adhesion inspontaneously hypertensive rats by adrenal corticosteroids. J. LeukocyteBiol. 57:20-26.

199. Fox, S. B., G. D. H. Turner, K. C. Gatter, and A. L. Harris. 1995.The increased expression of adhesion molecules ICAM-3, E- andP-selectins on breast cancer endothelium. J. Pathol. 177:369-376.

200. Borgström, P., G. K. Hughes, P. Hansell, B. A. Wolitzky, and P.Sriramarao. 1997. Leukocyte adhesion in angiogenic blood vessels—Role ofE-selectin, P-selectin, and β2 integrin in lymphotoxin-mediatedleukocyte recruitment in tumor microvessels. J. Clin. Invest.99:2246-2253.

We claim:
 1. A method of selectively inhibiting selectin mediatedbinding in a subject by administering an amount of heparin that does notproduce substantial anticoagulant activity or undesireable bleeding,wherein said selectin mediated binding is selected from the groupconsisting of L-selectin mediated binding and P-selectin mediatedbinding.
 2. The method of claim 1, wherein the amount of heparinadministered results in a concentration of about 0.05 to 0.4 units ofheparin/ml of plasma.
 3. The method of claim 1, wherein the amount ofheparin administered results in a concentration of about 0.05 to 0.2units of heparin/ml of plasma.
 4. The method of claim 1, wherein theamount of heparin administered results in a concentration of about 0.05to 0.1 units of heparin/ml of plasma.
 5. The method of claim 1, whereinP-selectin mediated binding is inhibited.
 6. The method of claim 5,wherein the amount of heparin administered results in a concentration ofabout 0.005 to 0.05 units of heparin/ml of plasma.
 7. The method ofclaim 5, wherein the amount of heparin administered results in aconcentration of about 0.005 to 0.02 units of heparin/ml of plasma.
 8. Amethod of inhibiting L-selectin mediated binding in a subject byadministering an amount of heparin that does not produce substantialanticoagulant activity or undesirable bleeding.
 9. The method of claim8, wherein the amount of heparin administered results in a concentrationof about 0.05 to 0.4 units of heparin/ml of plasma.
 10. The method ofclaim 8, wherein the amount of heparin administered results in aconcentration of about 0.05 to 0.2 units of heparin/ml of plasma. 11.The method of claim 8, wherein the amount of heparin administeredresults in a concentration of about 0.05 to 0.1 units of heparin/ml ofplasma.
 12. A method of inhibiting P-selectin mediated binding in asubject by administering an amount of heparin that does not producesubstantial anticoagulant activity or undesirable bleeding.
 13. Themethod of claim 12, wherein the amount of heparin administered resultsin a concentration of about 0.005 to 0.05 units of heparin/ml of plasma.14. The method of claim 12, wherein the amount of heparin administeredresults in a concentration of about 0.005 to 0.02 units of heparin/ml ofplasma.
 15. A method of treating L-selectin and P-selectin relatedpathology by administering an amount of heparin that does not producesubstantial anticoagulant activity or undesirable bleeding.
 16. Themethod of claim 15, wherein the pathology is selected from the groupconsisting of ischemia, reperfusion injury, acute inflammation, chronicinflammation, and cancer metastasis.
 17. A method of treating P-selectinrelated pathology by administering an amount of heparin that does notproduce substantial anticoagulant activity or undesirable bleeding. 18.The method of claim 17, wherein the pathology is selected from the groupconsisting of ischemia, reperfusion injury, acute inflammation, chronicinflammation, and cancer metastasis.
 19. The method of claim 1, whereinsaid administration is by injection.
 20. The method of claim 19, whereinsaid injection is selected from the group consisting of intravenous andsubcutaneous injection.
 21. The method of claim 8, wherein saidadministration is by injection.
 22. The method of claim 21, wherein saidinjection is selected from the group consisting of intravenous andsubcutaneous injection.
 23. The method of claim 15, wherein saidadministration is by injection.
 24. The method of claim 23, wherein saidinjection is selected from the group consisting of intravenous andsubcutaneous injection.
 25. The method of claim 17, wherein saidadministration is by injection.
 26. The method of claim 25, wherein saidinjection is selected from the group consisting of intravenous andsubcutaneous injection.